Method for producing butanol using extractive fermentation

ABSTRACT

Provided herein are methods for recovering butanol from a fermentation medium. The methods comprise providing a fermentation medium comprising butanol, water, and a recombinant microorganism comprising a butanol biosynthetic pathway, wherein the recombinant microorganism produces butanol; contacting the fermentation medium with a water immiscible organic extractant composition comprising a dry solvent to form a butanol-containing organic phase and an aqueous phase; and recovering the butanol from the butanol-containing organic phase.

CROSS-REFERENCE

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/211,342, filed on Mar. 14, 2014, which isrelated to and claims priority to U.S. Provisional Patent ApplicationNo. 61/790,828, filed on Mar. 15, 2013. Each application is herebyincorporated by reference in its entirety. Additionally, thisapplication incorporates by reference in their entireties U.S.Provisional Patent Application No. 61/788,213, filed on 15 Mar. 2013,entitled Method for Production of Butanol Using Extractive Fermentation,and U.S. Provisional Patent Application Nos. 61/790,401, filed on 15Mar. 2013, entitled Method for Production of Butanol Using ExtractiveFermentation.

FIELD OF THE INVENTION

The invention relates to the field of industrial microbiology and thefermentative production of butanol and isomers thereof. Morespecifically, the invention relates to a method for producing butanolthrough microbial fermentation, in which the butanol product is removedby extraction into a water-immiscible extractant composition whichcomprises a dry solvent.

BACKGROUND

Butanol is an important industrial chemical, useful as a fuel additive,as a feedstock chemical in the plastics industry, and as a food gradeextractant in the food and flavor industry. Each year 10 to 12 billionpounds of butanol are produced by petrochemical means and the need forthis commodity chemical will likely increase in the future.

Several chemical synthetic methods are known; however, these methods ofproducing butanol use starting materials derived from petrochemicals andare generally expensive and are not environmentally friendly. Severalmethods of producing butanol by fermentation are also known, for examplethe ABE process which is the fermentive process producing a mixture ofacetone, 1-butanol, and ethanol. Acetone-butanol-ethanol (ABE)fermentation by Clostridium acetobutylicum is one of the oldest knownindustrial fermentations; as are also the pathways and genes responsiblefor the production of these solvents. Production of 1-butanol by the ABEprocess is limited by the toxic effect of the 1-butanol on Clostridiumacetobutylicum. In situ extractive fermentation methods using specificextractants which are nontoxic to the bacterium have been reported toenhance the production of 1-butanol by fermentation using Clostridiumacetobutylicum (see, for example, Roffler et al., Biotechnol. Bioeng.31:135-143, 1998; Roffler et al., Bioprocess Engineering 2:1-12, 1987;and Evans et al., Appl. Environ. Microbiol. 54:1662-1667, 1988).

In contrast to the native Clostridium acetobutylicum described above,recombinant microbial production hosts expressing 1-butanol, 2-butanol,and isobutanol biosynthetic pathways have also been described. Theserecombinant hosts have the potential of producing butanol in higheryields compared to the ABE process because they do not producebyproducts such as acetone and ethanol. With these recombinant hosts,the biological production of butanol appears to be limited by thebutanol toxicity thresholds of the host microorganism used in thefermentation. U.S. Patent Publication No. 20090305370 discloses a methodof making butanol from at least on fermentable carbon source thatovercomes the issues of toxicity resulting in an increase in theeffective titer, the effective rate, and the effective yield of butanolproduction by fermentation utilizing a recombinant microbial hostwherein the butanol is extracted into specific organic extractantsduring fermentation.

Improved methods for producing and recovering butanol from afermentation medium are continually sought. Lower cost processes andimprovements to process operability are also desired. Identification ofimproved extractants for use with fermentation media, such asextractants exhibiting higher partition coefficients, lower viscosity,lower density, commercially useful boiling points, and sufficientmicrobial biocompatibility, is a continual need. Additionally,extractants that are selective for butanol over water provide certainadvantages. By way of an example, an extractant that is selective forbutanol over water can reduce the energy needs for a butanolfermentation process. The reduction in total energy needed to strip thebutanol from the extractant can be due to the reduction in waterassociated with the extractant, as the energy required to strip butanolfrom the extractant is directly related to the amount of water presentin the extractant.

The present invention satisfies the need to provide methods forrecovering butanol from a fermentation medium by contacting thefermentation medium with an organic extractant composition comprising adry solvent, wherein the dry solvent selectively extracts butanol fromthe fermentation medium.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods for recovering butanol from a fermentationmedium. The methods comprise (a) providing a fermentation mediumcomprising butanol, water, and a recombinant microorganism comprising abutanol biosynthetic pathway, wherein the recombinant microorganismproduces butanol; (b) contacting the fermentation medium with a waterimmiscible organic extractant composition comprising a dry solvent toform a butanol-containing organic phase and an aqueous phase; and (c)recovering the butanol from the butanol-containing organic phase.

In certain embodiments, the dry solvent is a saturated hydrocarbon. Thesaturated hydrocarbon can, for example, be a C₇ to C₂₂ alkane or amixture thereof. The C₇ to C₂₂ alkane can be a branched C₇ to C₂₂alkane. In some embodiments the alkane comprises up to a C₂₅ alkane. Infurther embodiments, the hydrocarbon is unsaturated or an aromatichydrocarbon. The hydrocarbon can, for example, be a derivative ofisobutanol. The derivative of isobutanol can, for example, betriisobutylene, diisobutylene, tetraisobutylene, isooctane,isohexadecane, 3,4,5,6,6-pentamethyl-2-heptanol, or isododecane.

In some embodiments, the organic extractant composition furthercomprises a second solvent. The second solvent can be, for example, a C₄to C₂₂ fatty alcohol, a C₄ to C₂₈ fatty acid, an ester of a C₄ to C₂₈fatty acid, a C₄ to C₂₂ fatty aldehyde, a C₇ to C₂₂ ether, amides,phosphate esters, ureas, phenols (phenolics), phosphinates, carbamates,phosphoramide, or mixtures thereof. The second solvent can be, forexample, oleyl alcohol, phenyl alcohol, cetyl alcohol, lauryl alcohol,myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristicacid, stearic acid, octanoic acid, decanoic acid, undecanoic acid,methyl myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol,1-nonanal, 1-undecanol, undecanal, isododecanol, lauric aldehyde,2-methylundecanal, oleamide, linoleamide, palmitamide, stearylamide,2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, or mixturesthereof. In embodiments, one or more of the solvents include one or moreof phosphorous, nitrogen, sulfur, or oxygen. For example, at least oneof the solvents is selected because it is polar or exhibits hydrogenbonding. The second solvent can increase the butanol partitioncoefficient of the organic extractant composition.

In some embodiments, the contacting of the organic extractantcomposition with the fermentation medium occurs in the fermentor. Inother embodiments, the contacting of the organic extractant compositionwith the fermentation medium occurs outside the fermentor. In someembodiments, the butanol is recovered after transferring a portion ofthe fermentation medium from the fermentor to a vessel, wherein thecontacting of the organic extractant composition with the fermentationmedium occurs in the vessel. In some embodiments, the butanol isisobutanol.

In some embodiments, the recovered butanol has an effective titer fromabout 20 g per liter to about 50 g per liter of the fermentation medium.In some embodiments, the recovered butanol has an effective titer fromabout 22 g per liter to about 80 g per liter. In some embodiments, therecovered butanol has an effective titer from about 25 g per liter toabout 50 g per liter. In embodiments, the recovered butanol has aneffective titer of at least 25 g, at least 30 g, at least 46 g, at least50 g, at least 60 g, or at least 70 g per liter of the fermentationmedium.

In some embodiments, the recovered butanol has an effective titer fromabout 20 g per liter to about 80 g per liter of the fermentation medium.In some embodiments, the recovered butanol has an effective titer fromabout 25 g per liter to about 50 g per liter. In some embodiments, therecovered butanol has an effective titer from about 30 g per liter toabout 80 g per liter. In embodiments, the recovered butanol has aneffective titer of at least 25 g, at least 30 g, at least 35 g, at least37 g, at least 40 g, or at least 45 g per liter of the fermentationmedium.

Also provided are compositions comprising butanol in a water immiscibleorganic extractant compositions, wherein the organic extractantcomposition comprises a solvent, wherein the solvent is a dry solvent.

In certain embodiments, the dry solvent is a saturated hydrocarbon. Thesaturated hydrocarbon can, for example, be a C₇ to C₂₂ alkane or amixture thereof. The C₇ to C₂₂ alkane can be a branched C₇ to C₂₂alkane. The hydrocarbon can, for example, be a derivative of isobutanol.The derivative of isobutanol can, for example, be triisobutylene,diisobutylene, tetraisobutylene, isooctane, isohexadecane,3,4,5,6,6-pentamethyl-2-heptanol, or isododecane.

In some embodiments, the organic extractant composition furthercomprises a second solvent. The second solvent can be, for example, a C₄to C₂₂ fatty alcohol, a C₄ to C₂₈ fatty acid, an ester of a C₄ to C₂₈fatty acid, a C₄ to C₂₂ fatty aldehyde, a C₇ to C₂₂ ether, or mixturesthereof. The second solvent can be, for example, oleyl alcohol, phenylalcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearylalcohol, oleic acid, lauric acid, myristic acid, stearic acid, octanoicacid, decanoic acid, undecanoic acid, methyl myristate, methyl oleate,1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, undecanol, undecanal,isododecanol, lauric aldehyde, 2-methylundecanal, oleamide, linoleamide,palmitamide, stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol,2-octyl-1-dodecanol, or mixtures thereof. Additional examples include,phosphine oxides, tetraalkyureas, alkylphenol, parabens, salicylates,and so forth. The second solvent can increase the butanol partitioncoefficient of the organic extractant composition.

In some embodiments, the butanol is isobutanol.

In some embodiments, a method of extracting alcohol from an aqueoussolution comprises (A) selecting which solvents to be included in asolvent mixture by identifying a first solvent and a second solventbased on the first and second solvents' respective properties, whereinthe first and second solvents have a similar chemical structure; (B)setting a limit for a ratio of the first and second solvents to beincluded in the solvent mixture based on the solvent mixture'shydrophobicity, where the hydrophobicity of the solvent mixture is notindicated by a linear combination of the first solvent's hydrophobicityand the second solvents hydrophobicity; (C) determining a ratio of thefirst solvent to the second solvent to be included in the solventmixture within the limit to balance the solvent mixture's overallproperties so the solvent mixture exhibits at least one synergisticproperty that is not indicated by a linear combination of the first andsecond solvents, respective, properties that correspond to the at leastone synergistic property; and (D) contacting an aqueous solutionincluding the alcohol with the solvent mixture to extract the alcohol.In examples, the hydrophobicity of the first solvent is the base ten logof the ratio of an amount first solvent in an organic phase divided byan amount of the first solvent in aqueous phase for a tertiary mixtureof the first solvent and water in the presence of octanol; wherein thehydrophobicity of the second solvent is the base ten log of the ratio ofan amount second solvent in an organic phase divided by an amount of thesecond solvent in aqueous phase for a tertiary mixture of the secondsolvent and water in the presence of octanol; and wherein thehydrophobicity of the solvent mixture is the base ten log of the ratioof an amount of solvent mixture in an organic phase divided by an amountof the solvent mixture in aqueous phase for a mixture of the solventmixture and water in the presence of octanol. In additional examples,hydrophobicity comprises an indicator of biocompatibility. In furtherembodiments, the method includes interating steps A, B, and C for eachsolvent to be included in the solvent mixture to account for the solventmixture's cumulative properties based on the first solvent's properties,the second solvent's properties, and the each solvent that isadditionally included in the solvent mixture. In an example, the atleast one synergistic property comprises hydrophobicity. In additionalexamples, hydrophobicity is expressed as log P. In embodiments, the atleast one synergistic property comprises the solvent mixture's abilityto solubilize water. In some examples, the at least one synergisticproperty comprises a partition coefficient of the solvent mixture in amix with butanol and water (Kd). In some embodiments, the at least onesynergistic property comprises biocompatibility with a microorganismthat produces the alcohol. Additionally, in an example, themicroorganism comprises a genetically modified microorganism. In furtherembodiments, the genetically modified microorganism comprises abutanologen. In some embodiments, at least one of the first or secondsolvents is substantially biocompatible with the butanologen.Additionally, in some embodiments, the first solvent is comparativelymore bio-incompatible with the butanologen than the second solvent andexhibits greater alcohol selectivity than the second solvent. In someembodiments, at least one synergistic property comprises a low affinityto extract a nutrient from the aqueous solution. In examples, thenutrient comprise a nutrient that supports alcohol fermentation by amicroorganism. In further examples, the butanologen comprises abutanologen with a biosynthetic pathway engineered to yield butanol inhigh amount in comparison to the ABE process. In some embodiments, theaqueous solution comprises fermentation broth including a microorganismgenetically modified to produce the alcohol. In examples, the alcoholcomprises a fusel. In some examples, the first solvent comprises aderivative of isobutanol. Further, in examples, the derivative ofisobutanol comprises at least one of triisobutylene, diisobutylene,tetraisobutylene, isooctane, isohexadecane,3,4,5,6,6-pentamethyl-2-heptanol, or isododecane. In embodiments inaccordance with this disclosure, the first solvent comprises at leastone of a C4 to C22 fatty alcohol, a C4 to C28 fatty acid, an ester of aC4 to C28 fatty acid, a C4 to C22 fatty aldehyde, a C7 to C22 ether,amides, phosphate esters, ureas, phenols (phenolics), phosphinates,carbamates, phosphoramide, or mixtures thereof. The first solventcomprises at least one of oleyl alcohol, phenyl alcohol, cetyl alcohol,lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauricacid, myristic acid, stearic acid, octanoic acid, decanoic acid,undecanoic acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol,2-undecanol, 1-nonanal, 1-undecanol, undecanal, isododecanol, lauricaldehyde, 2-methylundecanal, oleamide, linoleamide, palmitamide,stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol,or mixtures thereof in embodiments of the present disclosure.

In some embodiments, a method of extracting alcohol from an aqueoussolution comprises combining at least two solvents to form a solventmixture that exhibits a synergistic alcohol extraction property that isnot indicated by a linear combination of properties, where respectiveones of the properties correspond to the at least two solvents, the atleast two solvents being selected by identifying the at least twosolvents based on the respective ones of the properties that correspondto the at least two solvents, wherein two of the at least two solventshave corresponding chemical structures; setting a limit for a ratio ofthe at least two solvents relative to each solvent in the solventmixture based on each solvent's hydrophobicity, where the solventmixture's hydrophobicity is not indicated by a linear combination of thehydrophobicities for each of the solvent included in the solventmixture; and mixing the at least two solvents in a ratio within thelimit to balance the solvent mixture's overall properties so the solventmixture exhibits the synergistic alcohol extraction property. Inembodiments, the method further comprises contacting an aqueous solutionthat includes the alcohol with the solvent mixture to extract thealcohol. In examples, the synergistic alcohol extraction propertycomprises the solvent mixture's ability to solubilize water. Inadditional examples, hydrophobicity comprises a partition coefficient ofat least one solvent included in the at least two solvents or thesolvent mixture in a mix with octanol, and water (log P). In furtherexamples, the synergistic alcohol extraction property comprises apartition coefficient of the solvent mixture in a mix with butanol, andwater (Kd). In an example, at least one solvent in the solvent mixturecomprises a dry solvent that exhibits good biocompatibility with amicroorganism capable of producing the alcohol and at least one solventin the solvent mixture comprises a solvent that exhibits high affinityto the alcohol. In additional examples, the alcohol comprises a fusel.In examples, the fusel comprises butanol. Further examples include wherethe butanol comprises isobutanol. In embodiments, the aqueous solutioncomprises a broth that includes an isobutanolgen that is geneticallymodified to yield more isobutanol in comparison to the ABE process. Insome embodiments, the synergistic alcohol extraction property comprisesa poor extraction efficiency to a nutrient consumed by a microorganismto produce the alcohol. Further examples exist where hydrophobicitycomprises an indicator of biocompatibility. In embodiments, the limit isapproximate a log P of six (6). In some examples, the limit correspondsto a concentration of solvent mixture in the aqueous solution that isinsufficient to appreciably impact integrity of a microorganism's cellmembrane.

In some embodiments, a method of drying an extractant comprisescontacting, with a first solvent, a fermentation broth that includes arecombinant microorganism comprising a butanol biosynthetic pathway andbutanol produced via that butanol biosynthetic pathway to extract atleast a portion of the butanol into the first solvent; and contactingthe first solvent that includes at least a portion of the butanol and atleast some water from the fermentation broth with a second solvent toextract the water from the first solvent into the second solvent to drythe first solvent including the butanol. In methods in accordance withthese embodiments contacting the first and second solvents is performedout of the fermentation broth's presence. In further examples, the firstsolvent comprises a solvent mixture that is prepared by: combining atleast two solvents to form the solvent mixture that exhibits asynergistic alcohol extraction property that is not indicated by alinear combination of properties, where respective ones of theproperties correspond to the at least two solvents, the at least twosolvents being selected by: identifying the at least two solvents basedon the respective ones of the properties that correspond to the at leasttwo solvents, wherein two of the at least two solvents havecorresponding chemical structures; setting a limit for a ratio of the atleast two solvents relative to each solvent in the solvent mixture basedon each solvent's hydrophobicity, where the solvent mixture'shydrophobicity is not indicated by a linear combination of thehydrophobicities for each of the solvent included in the solventmixture; and mixing the at least two solvents in a ratio within thelimit to balance the solvent mixture's overall properties so the solventmixture exhibits the synergistic alcohol extraction property. In someexamples, the synergistic alcohol extraction property comprises thesolvent mixture's ability to reject water. In some embodiments,hydrophobicity comprises a partition coefficient of at least one solventincluded in the at least two solvents or the solvent mixture in a mixwith octanol, and water (log P). Additionally, examples exist where thesynergistic alcohol extraction property comprises a partitioncoefficient of the solvent mixture in a mix with butanol, and water(Kd). Further in an example, at least one solvent in the solvent mixturecomprises a dry solvent that exhibits good biocompatibility with amicroorganism capable of producing the alcohol and at least one solventin the solvent mixture comprises a solvent that exhibits high affinityto the alcohol. In further embodiments, the alcohol comprises a fusel.Moreover, in some examples, the fusel comprises butanol. In furtherexamples, the butanol comprises isobutanol. In some examples, thesynergistic alcohol extraction property comprises a poor extractionefficiency to a nutrient consumed by a microorganism to produce thealcohol. In additional embodiments, hydrophobicity comprises anindicator of biocompatibility. In some embodiments in accordance withthis disclosure, the limit is approximately log P of six (6). The methodcan also include where the limit corresponds to a concentration ofsolvent mixture in the aqueous solution that is insufficient toappreciably impact integrity of a microorganism's cell membrane. Inexamples, the second solvent comprises glycerol. Additionally, inexamples contacting the first and second solvents is performed as acountercurrent extraction that is performed after the first solvent iscontacted with the fermentation broth. In embodiments in accordance withthe present disclosure the first solvent comprises a dry solvent.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 schematically illustrates one embodiment of the methods of theinvention, in which the first water immiscible extractant and theoptional second water immiscible extractant are combined in a vesselprior to contacting the fermentation medium with the extractant in afermentation vessel.

FIG. 2 schematically illustrates one embodiment of the methods of theinvention, in which the first water immiscible extractant and theoptional second water immiscible extractant are added separately to afermentation vessel in which the fermentation medium is contacted withthe extractant.

FIG. 3 schematically illustrates one embodiment of the methods of theinvention, in which the first water immiscible extractant and theoptional second water immiscible extractant are added separately todifferent fermentation vessels for contacting of the fermentation mediumwith the extractant.

FIG. 4 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs downstream of thefermentor and the first water immiscible extractant and the optionalsecond water immiscible extractant are combined in a vessel prior tocontacting the fermentation medium with the extractant in a differentvessel.

FIG. 5 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs downstream of thefermentor and the first water immiscible extractant and the optionalsecond water immiscible extractant are added separately to a vessel inwhich the fermentation medium is contacted with the extractant.

FIG. 6 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs downstream of thefermentor and the first water immiscible extractant and the optionalsecond water immiscible extractant are added separately to differentvessels for contacting of the fermentation medium with the extractant.

FIG. 7 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs in at leaston-batch fermentor via co-current flow of a water-immiscible extractantcomprising a first solvent and an optional second solvent at or near thebottom of a fermentation mash to fill the fermentor with extractantwhich flows out of the fermentor at a point at or near the top of thefermentor.

FIG. 8 shows a schematic of a process for converting isobutanol toderivatives of isobutanol, e.g., triisobutylene, diisobutylene,tetraisobutylene, and isododecane.

FIG. 9 is a graph showing the relationship between water content andheat requirement to reach a preheater exit temperature.

FIG. 10 is a graph showing the relationship between the water contentand heat requirement to reach a heat exchanger temperature of 100° C.

FIG. 11A is a graphical illustration of model estimations for COFA andisododecane in comparison to a linear combination model of COFA andisododecane.

FIG. 11B graphically illustrates how various molar concentrations ofisododecane/isododecanol impact Kd for the solvent mixture.

FIG. 11C graphically illustrates how various molar concentrations oftetrabutylurea with isododecane impact Kd for the solvent mixture.

FIG. 12 is a graphical illustration of the Kd properties (partitioncoefficient for butanol) of several alkyphenol solvents relative to logP (hydrophobicity) for the solvents.

FIG. 13 is a graphical illustration of boiling points relative to log P(hydrophobicity) of several solvents including notations relating to thenumber of carbon atoms in the solvents.

FIG. 14, is a graphical illustration of log P versus molarconcentrations for solvent mixtures of corn oil and thymol and mixturesof triisopropylbenzene and thymol.

FIG. 15, is a graphical illustration of expected versus PotentialObserved Properties.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference, unless only specific sections of patents orpatent publications are indicated to be incorporated by reference.Headings are implemented throughout this document to aid the reader'sunderstanding of the disclosed subject matter. These headings areprovided solely for the reader's convenience and should not beconsidered as limiting or dividing this disclosure into parts. And, thetechniques, approaches, methodologies, systems and devices described inconjunction with one portion are generally applicable to other portionsof this disclosure.

In order to further define this invention, the following terms,abbreviations and definitions are provided.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers can be added to thespecified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition. See M.P.E.P. §2111.03.

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances, i.e., occurrences of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the claims as presented or as later amended andsupplemented, or in the specification.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates orsolutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or to carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, or within 5% of the reported numerical value.

The term “butanol biosynthetic pathway” as used herein refers to theenzymatic pathway to produce 1-butanol, 2-butanol, or isobutanol.

The term “1-butanol biosynthetic pathway” refers to an enzymatic pathwayto produce 1-butanol. A “1-butanol biosynthetic pathway” can refer to anenzyme pathway to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA).For example, 1-butanol biosynthetic pathways are disclosed in U.S.Patent Application Publication No. 2008/0182308 and InternationalPublication No. WO 2007/041269, which are herein incorporated byreference in their entireties.

The term “2-butanol biosynthetic pathway” refers to an enzymatic pathwayto produce 2-butanol. A “2-butnaol biosynthetic pathway” can refer to anenzyme pathway to produce 2-butanol from pyruvate. For example,2-butanol biosynthetic pathways are disclosed in U.S. Pat. No.8,206,970, U.S. Patent Application Publication No. 2007/0292927,International Publication Nos. WO 2007/130518 and WO 2007/130521, whichare herein incorporated by reference in their entireties.

The term “isobutanol biosynthetic pathway” refers to an enzymaticpathway to produce isobutanol. An “isobutanol biosynthetic pathway” canrefer to an enzyme pathway to produce isobutanol from pyruvate. Forexample, isobutanol biosynthetic pathways are disclosed in U.S. Pat. No.7,851,188, U.S. Application Publication No. 2007/0092957, andInternational Publication No. WO 2007/050671, which are hereinincorporated by reference in their entireties. From time to time“isobutanol biosynthetic pathway” is used synonymously with “isobutanolproduction pathway.”

The term “butanol” as used herein refers to the butanol isomers1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol (t-BuOH), and/orisobutanol (iBuOH or i-BuOH, also known as 2-methyl-1-propanol), eitherindividually or as mixtures thereof. From time to time, as used hereinthe terms “biobutanol” and “bio-produced butanol” may be usedsynonymously with “butanol.”

Uses for butanol can include, but are not limited to, fuels (e.g.,biofuels), a fuel additive, an alcohol used for the production of estersthat can be used as diesel or biodiesel fuel, as a chemical in theplastics industry, an ingredient in formulated products such ascosmetics, and a chemical intermediate. Butanol may also be used as asolvent for paints, coatings, varnishes, resins, gums, dyes, fats,waxes, resins, shellac, rubbers, and alkaloids.

As used herein, the term “bio-produced” means that the molecule (e.g.,butanol) is produced from a renewable source (e.g., the molecule can beproduced during a fermentation process from a renewable feedstock).Thus, for example, bio-produced isobutanol can be isobutanol produced bya fermentation process from a renewable feedstock. Molecules producedfrom a renewable source can further be defined by the ¹⁴C/¹²C isotoperatio. A ¹⁴C/¹²C isotope ratio in range of from 1:0 to greater than 0:1indicates a bio-produced molecule, whereas a ratio of 0:1 indicates thatthe molecule is fossil derived.

“Product alcohol” as used herein, refers to any alcohol that can beproduced by a microorganism in a fermentation process that utilizesbiomass as a source of fermentable carbon substrate. Product alcoholsinclude, but are not limited to, C₁ to C₈ alkyl alcohols, and mixturesthereof. In some embodiments, the product alcohols are C₂ to C₈ alkylalcohols. In other embodiments, the product alcohols are C₂ to C₅ alkylalcohols. It will be appreciated that C₁ to C₈ alkyl alcohols include,but are not limited to, methanol, ethanol, propanol, butanol, pentanol,and mixtures thereof. Likewise C₂ to C₈ alkyl alcohols include, but arenot limited to, ethanol, propanol, butanol, and pentanol. “Alcohol” isalso used herein with reference to a product alcohol.

A recombinant host cell comprising an “engineered alcohol productionpathway” (such as an engineered butanol or isobutanol productionpathway) refers to a host cell containing a modified pathway thatproduces alcohol in a manner different than that normally present in thehost cell. Such differences include production of an alcohol nottypically produced by the host cell, or increased or more efficientproduction.

The term “heterologous biosynthetic pathway” as used herein refers to anenzyme pathway to produce a product in which at least one of the enzymesis not endogenous to the host cell containing the biosynthetic pathway.

The term “extractant” as used herein refers to one or more organicsolvents which can be used to extract a product alcohol. From time totime as used herein, the term “extractant” may be used synonymously with“solvent.”

The term “dry solvent” as used herein refers to a solvent thatselectively extracts the product alcohol (e.g., isobutanol) from anaqueous medium over water. By way of an example, a dry solvent canextract the product alcohol over water such that the equilibrium watercontent in the solvent is less than about 0.05%.

The term “effective isobutanol productivity” as used herein refers tothe total amount in grams of isobutanol produced per gram of cells.

The term “effective titer” as used herein, refers to the total amount ofa particular alcohol (e.g., butanol) produced by fermentation per literof fermentation medium. The total amount of butanol includes: (i) theamount of butanol in the fermentation medium; (ii) the amount of butanolrecovered from the organic extractant; and (iii) the amount of butanolrecovered from the gas phase, if gas stripping is used.

The term “effective rate” as used herein, refers to the total amount ofbutanol produced by fermentation per liter of fermentation medium perhour of fermentation.

The term “effective yield” as used herein, refers to the amount ofbutanol produced per unit of fermentable carbon substrate consumed bythe biocatalyst.

The term “separation” as used herein is synonymous with “recovery” andrefers to removing a chemical compound from an initial mixture to obtainthe compound in greater purity or at a higher concentration than thepurity or concentration of the compound in the initial mixture.

The term “In Situ Product Removal” (ISPR) as used herein refers to theselective removal of a fermentation product from a biological processsuch as fermentation to control the product concentration as the productis produced.

The term “aqueous phase,” as used herein, refers to the aqueous phase ofa biphasic mixture obtained by contacting a fermentation broth with awater-immiscible organic extractant. In an embodiment of a processdescribed herein that includes fermentative extraction, the term“fermentation broth” then specifically refers to the aqueous phase inbiphasic fermentative extraction, and the terms “solvent-poor phase” maybe used synonymously with “aqueous phase” and “fermentation broth.”.

The term “organic phase,” as used herein, refers to the non-aqueousphase of a biphasic mixture obtained by contacting a fermentation brothwith a water-immiscible organic extractant. From time to time, as usedherein the terms “solvent-rich phase” may be used synonymously with“organic phase.”

The term “aqueous phase titer” as used herein, refers to theconcentration of product alcohol (e.g., butanol) in the fermentationbroth.

The term “water-immiscible” as used herein refers to a chemicalcomponent such as an extractant or a solvent, which is incapable ofmixing with an aqueous solution such as a fermentation broth, in such amanner as to form one liquid phase.

The term “moisture content” as used herein refers to the equilibriumsaturation of water contained by the solvent, whether or not productalcohol, e.g., isobutanol is present. At times the term “equilibrium” isused in conjunction with “moisture content” to indicate “moisturecontent” is related to a particular set of conditions, e.g.,temperature, pressure, and so on. As will, be apparent, moisture contentcommonly refers to a maximum amount of water that can be dissolve by thesolvent/solvent mixture given a certain set of conditions.

The term “biphasic fermentation medium” as used herein refers to atwo-phase growth medium comprising a fermentation medium (i.e., anaqueous phase) and a suitable amount of a water-immiscible organicextractant.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof. Non-limiting examples ofcarbon substrates are provided herein and include, but are not limitedto, monosaccharides, disaccharides, oligosaccharides, polysaccharides,ethanol, lactate, succinate, glycerol, carbon dioxide, methanol,glucose, fructose, lactose, sucrose, xylose, arabinose, dextrose,cellulose, methane, amino acids, or mixtures thereof.

“Fermentation broth” as used herein means the mixture of water, sugars(fermentable carbon sources), dissolved solids (if present),microorganisms producing alcohol, product alcohol and all otherconstituents of the material in which product alcohol is being made bythe reaction of sugars to alcohol, water and carbon dioxide (CO₂) by themicroorganisms present. From time to time, as used herein the term“fermentation medium” and “fermented mixture” can be used synonymouslywith “fermentation broth.”

As used herein a “fermentor” refers to any container, containers, orapparatus that are used to ferment a substrate. A fermentor can containa fermentation medium and microorganism capable of fermentation. Theterm “fermentation vessel” refers to the vessel in which thefermentation reaction is carried out whereby alcohol such as butanol ismade from sugars. “Fermentor” can be used herein interchangeable with“fermentation vessel.”

The term “fermentation product” includes any desired product ofinterest, including, but not limited to 1-butanol, 2-butanol,isobutanol, etc.

The term “sugar” as used herein, refers to oligosaccharides,disaccharides, monosaccharides, and/or mixtures thereof. The term“saccharide” also includes carbohydrates including starches, dextrans,glycogens, cellulose, pentosans, as well as sugars.

The term “fermentable sugar” as used herein, refers to one or moresugars capable of being metabolized by the microorganisms disclosedherein for the production of fermentative alcohol.

The term “undissolved solids” as used herein, means non-fermentableportions of feedstock, for example, germ, fiber, and gluten. Forexample, the non-fermentable portions of feedstock include the portionof feedstock that remains as solids and can absorb liquid from thefermentation broth.

“Biomass” as used herein refers to a natural product containing ahydrolysable starch that provides a fermentable sugar, including anycellulosic or lignocellulosic material and materials comprisingcellulose, and optionally further comprising hemicellulose, lignin,starch, oligosaccharides, disaccharides, and/or monosaccharides. Biomasscan also comprise additional components, such as protein and/or lipids.Biomass can be derived from a single source, or biomass can comprise amixture derived from more than one source. For example, biomass cancomprise a mixture of corn cobs and corn stover, or a mixture of grassand leaves. Biomass includes, but is not limited to, bioenergy crops,agricultural residues, municipal solid waste, industrial solid waste,sludge from paper manufacture, yard waste, wood, and forestry waste.Examples of biomass include, but are not limited to, corn grain, corncobs, crop residues such as corn husks, corn stover, grasses, wheat,rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass,waste paper, sugar cane bagasse, sorghum, soy, components obtained frommilling of grains, trees, branches, roots, leaves, wood chips, sawdust,shrubs and bushes, vegetables, fruits, flowers, animal manure, andmixtures thereof.

“Feedstock” as used herein means a product containing a fermentablecarbon source. Suitable feedstock include, but are not limited to, rye,wheat, corn, corn mash, cane, cane mash, sugar cane, barley, cellulosicmaterial, lignocellulosic material, and mixtures thereof.

The term “aerobic conditions” as used herein means growth conditions inthe presence of oxygen.

The term “microaerobic conditions” as used herein means growthconditions with low levels of oxygen (i.e., below normal atmosphericoxygen levels).

The term “anaerobic conditions” as used herein means growth conditionsin the absence of oxygen.

The term “minimal media” as used herein refers to growth media thatcontain the minimum nutrients possible for growth, generally without thepresence of amino acids. A minimal medium typically contains afermentable carbon source and various salts, which may vary amongmicroorganisms and growing conditions; these salts generally provideessential elements such as magnesium, nitrogen, phosphorous, and sulfurto allow the microorganism to synthesize proteins and nucleic acids.

The term “defined media” as used herein refers to growth media that haveknown quantities of all ingredients, e.g., a defined carbon source andnitrogen source, and trace elements and vitamins required by themicroorganism.

The term “biocompatibility” as used herein refers to the measure of theability of a microorganism to utilize glucose in the presence of anextractant. A biocompatible extractant permits the microorganism toutilize glucose. A non-biocompatible (i.e., a biotoxic) extractant doesnot permit the microorganism to utilize glucose, for example, at a rategreater than about 25% of the rate when the extractant is not present.

The term “toxicity” of solvent as used herein refers to the percentageof butanol-producing microorganisms killed after exposure to the solventfor a prolonged time, for example 24 hours.

The term “free volume” as used herein refers to the proportion of avolume of bulk solvent that is not occupied by solvent molecules.

The term “fatty acid” as used herein, refers to a carboxylic acid (e.g.,aliphatic monocarboxylic acid) having C₄ to C₂₈ carbon atoms (mostcommonly C₁₂ to C₂₄ carbon atoms), which is either saturated orunsaturated. Fatty acids may also be branched or unbranched. Fatty acidsmay be derived from, or contained in esterified form, in an animal orvegetable fat, oil, or wax. Fatty acids may occur naturally in the formof glycerides in fats and fatty oils or may be obtained by hydrolysis offats or by synthesis. The term fatty acid may describe a single chemicalspecies or a mixture of fatty acids. In addition, the term fatty acidalso encompasses free fatty acids.

The term “fatty alcohol” as used herein, refers to an alcohol having analiphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “fatty aldehyde” as used herein, refers to an aldehyde havingan aliphatic chain of C₄ to C₂₂ carbon atoms, which is either saturatedor unsaturated.

The term “fatty amide” as used herein, refers to an amide having a long,aliphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “fatty ester” as used herein, refers to an ester having a longaliphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “carboxylic acid” as used herein, refers to any organiccompound with the general chemical formula —COOH in which a carbon atomis bonded to an oxygen atom by a double bond to make a carbonyl group(—C═O) and to a hydroxyl group (—OH) by a single bond. A carboxylic acidmay be in the form of the protonated carboxylic acid, in the form of asalt of a carboxylic acid (e.g., an ammonium, sodium, or potassiumsalt), or as a mixture of protonated carboxylic acid and salt of acarboxylic acid. The term carboxylic acid may describe a single chemicalspecies (e.g., oleic acid) or a mixture of carboxylic acids as can beproduced, for example, by the hydrolysis of biomass-derived fatty acidesters or triglycerides, diglycerides, monoglycerides, andphospholipids.

The term “hydrocarbon” as used herein refers to a molecule that containshydrogen and carbon atoms.

The term “alkane” as used herein refers to a saturated hydrocarbon.

The term “alkene” as used herein refers to an unsaturated hydrocarboncontaining at least one carbon to carbon double bond.

The term “branched alkane” as used herein refers to an alkane with alkylside groups.

The term “isododecane” as used herein refers to an alkane with thelongest straight carbon chain of seven. “Isododecane” can also bereferred to as “pentamethyl heptane.”

“Portion” as used herein, includes a part of a whole or the whole. Forexample, a portion of fermentation broth includes a part of thefermentation broth as well as the whole (or all) the fermentation broth.

“Partition coefficient” refers to the ratio of the concentration of acompound in the two phases of a mixture of two immiscible solvents atequilibrium. A partition coefficient is a measure of the differentialsolubility of a compound between two immiscible solvents. Partitioncoefficient, as used herein, is synonymous with the term distributioncoefficient.

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene can comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome of amicroorganism. A “foreign” gene refers to a gene not normally found inthe host microorganism, but that is introduced into the hostmicroorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native microorganism, or chimeric genes. A“transgene” is a gene that has been introduced into the genome by atransformation procedure.

As used herein, “native” refers to the form of a polynucleotide, gene,or polypeptide as found in nature with its own regulatory sequences, ifpresent.

As used herein the term “coding sequence” or “coding region” refers to aDNA sequence that encodes for a specific amino acid sequence.

As used herein, “endogenous” refers to the native form of apolynucleotide, gene or polypeptide in its natural location in theorganism or in the genome of an organism. “Endogenous polynucleotide”includes a native polynucleotide in its natural location in the genomeof an organism. “Endogenous gene” includes a native gene in its naturallocation in the genome of an organism. “Endogenous polypeptide” includesa native polypeptide in its natural location in the organism transcribedand translated from a native polynucleotide or gene in its naturallocation in the genome of an organism.

The term “heterologous” when used in reference to a polynucleotide, agene, or a polypeptide refers to a polynucleotide, gene, or polypeptidenot normally found in the host organism. “Heterologous” also includes anative coding region, or portion thereof, that is reintroduced into thesource organism in a form that is different from the correspondingnative gene, e.g., not in its natural location in the organism's genome.The heterologous polynucleotide or gene can be introduced into the hostorganism by, e.g., gene transfer. A heterologous gene can include anative coding region with non-native regulatory regions that isreintroduced into the native host. For example, a heterologous gene caninclude a native coding region that is a portion of a chimeric geneincluding non-native regulatory regions that is reintroduced into thenative host. “Heterologous polypeptide” includes a native polypeptidethat is reintroduced into the source organism in a form that isdifferent from the corresponding native polypeptide. A “heterologous”polypeptide or polynucleotide can also include an engineered polypeptideor polynucleotide that comprises a difference from the “native”polypeptide or polynucleotide, e.g., a point mutation within theendogenous polynucleotide can result in the production of a“heterologous” polypeptide. As used herein a “chimeric gene,” a “foreigngene,” and a “transgene,” can all be examples of “heterologous” genes.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure.

Organic Extractants

A product alcohol may be recovered from fermentation broth using anumber of methods including liquid-liquid extraction. In someembodiments of the processes and systems described herein, an extractantmay be used to recover product alcohol from fermentation broth.Extractants used herein may be have, for example, one or more of thefollowing properties and/or characteristics: (i) biocompatible with themicroorganisms, (ii) immiscible with the fermentation medium, (iii) ahigh partition coefficient (K_(d)) for the extraction of productalcohol, (iv) a low partition coefficient for the extraction ofnutrients and other side products, (v) a low spreading coefficient, (vi)a high interfacial tension with water, (vii) low viscosity (μ), (viii)high selectivity for product alcohol as compared to, for example, water,(ix) low density (ρ) relative to the fermentation medium, (x) boilingpoint suitable for downstream processing of the extractant and productalcohol, (xi) melting point lower than ambient temperature, (xii)minimal solubility in solids, (xiii) a low tendency to form emulsionswith the fermentation medium, (xiv) stability over the fermentationprocess, (xv) low cost, (xvi) commercial availability, and (xvii)nonhazardous.

In some embodiments, the extractant may be selected based upon certainproperties and/or characteristics as described above. For example,viscosity of the extractant can influence the mass transfer propertiesof the system, i.e., the efficiency with which the product alcohol maybe extracted from the aqueous phase to the extractant phase (i.e.,organic phase). The density of the extractant can affect phaseseparation. In some embodiments, the extractant may be liquid at thetemperatures of the fermentation process. In some embodiments,selectivity refers to the relative amounts of product alcohol to watertaken up by the extractant. The boiling point can affect the cost andmethod of product alcohol recovery. For example, in the case wherebutanol is recovered from the extractant phase by distillation, theboiling point of the extractant should be sufficiently low as to enableseparation of butanol using available steam while minimizing any thermaldegradation or side reactions of the extractant, or the need for vacuumin the distillation process.

The extractant can be biocompatible with the microorganism, that is,nontoxic to the microorganism or toxic only to such an extent that themicroorganism is impaired to an acceptable level. In some embodiments,biocompatible refers to the measure of the ability of a microorganism toutilize fermentable carbon sources in the presence of an extractant. Theextent of biocompatibility of an extractant may be determined, forexample, by the glucose utilization rate of the microorganism in thepresence of the extractant and product alcohol. In some embodiments, anon-biocompatible extractant refers to an extractant that interfereswith the ability of a microorganism to utilize fermentable carbonsources. For example, a non-biocompatible extractant does not permit themicroorganism to utilize glucose at a rate greater than about 25%,greater than about 30%, greater than about 35%, greater than about 40%,greater than about 45%, or greater than about 50% of the rate when theextractant is not present.

One skilled in the art may select an extractant to maximize the desiredproperties and/or characteristics as described above and to optimizerecovery of a product alcohol. One of skill in the art can alsoappreciate that it may be advantageous to use a mixture of extractants.For example, extractant mixtures may be used to increase the partitioncoefficient for the product alcohol. Additionally, extractant mixturesmay be used to adjust and optimize physical characteristics of theextractant, such as the density, boiling point, and viscosity. Forexample, the appropriate combination may provide an extractant which hasa sufficient affinity for the product alcohol (Kd for butanol),hydrophobicity (log P), sufficient biocompatibility to enable itseconomical use for removing product alcohol from a fermentative broth(hydrophobicity expressed as log P can indicate biocompatibility),moisture content (tendency to solubilize water) and sufficientselectivity to enable the selective removal of the product alcohol over,for example, water.

In some embodiments, extractants useful in the processes and systemsdescribed herein may be organic solvents. In some embodiments, theextractants useful in the processes and systems described herein may bedry solvents. Dry solvents can, for example, be advantageous byattracting butanol and for providing little or no affinity to water. Adry solvent that offers no hydrogen bonding to water, for example, canabsorb the alcohol selectively. In some embodiments, the dry solventsmay comprise C₇ to C₂₂ hydrocarbons. In some embodiments, the drysolvents may comprise C₇ to C₂₂ alkanes or mixtures thereof. In someembodiments, the dry solvents may comprise C₇ to C₂₂ alkenes or mixturesthereof. The C₇ to C₂₂ alkanes or alkenes can, for example, be branchedalkanes or alkenes (e.g., the alkanes or alkenes may comprise alkyl sidegroups such as a methyl, an ethyl, a propyl, a butyl, a pentyl, or ahexyl side group). In some embodiments the hydrocarbons can bederivatives of isobutanol. The derivatives of isobutanol can, forexample, be selected from triisobutylene, isododecane, diisobutylene,tetraisobutylene, isooctane, 3,4,5,6,6-pentamethyl-2-heptanol, orisohexadecane.

Another advantage of dry solvents is the lower viscosity, higherinterfacial tension, and higher thermal and chemical stability that aidsin the phase separability and long term reuse. In some embodiments, adry solvent that accommodates the alkyl portion of butanol may becombined with another extractant that offers affinity in the form ofhydrogen bonding, for example, to the hydroxyl portion of butanol suchthat the mixture provides an optimal balance between selectivity andpartitioning over water. Advantages of saturated alkanes include a highinterfacial tension, higher thermal and chemical stability, goodbiocompatibility, a lower melting point, a lower boiling point, a lowdensity, a low viscosity, and a low tendency to form emulsions. Anotheradvantage with regard to higher partition coefficients, withoutintending to be limited by theory, some examples indicate that solventswith hydrogen bonding characteristics and/or high free volume have ahigh butanol partition coefficient (K_(d)). Increased hydrogen bondingcharacteristics can be achieved by having a greater number of hydrogenbonding sites per molecule. In some embodiments, compounds includingnitrogen, oxygen, phosphorus, and sulfur are used to provide hydrogenbonding sites. Free volume in the organic phase can be achieved usingsolvents whose molecules have a high degree of branching and do not packclosely.

In some embodiments, the organic extractant composition furthercomprises a second solvent. The second solvent can, for example, be anorganic solvent selected from the group consisting of saturated,mono-unsaturated, polyunsaturated, branched (and mixtures thereof) C₁₂to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides, C₇ toC₂₂ ethers, and mixtures thereof. The second solvent may also be anorganic solvent selected from the group consisting of saturated,mono-unsaturated, poly-unsaturated, branched (and mixtures thereof) C₄to C₂₂ fatty alcohols, C₄ to C₂₈ fatty acids, esters of C₄ to C₂₈ fattyacids, C₄ to C₂₂ fatty aldehydes, and mixtures thereof. In someembodiments, the extractant may include a first dry solvent and a secondsolvent selected from C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids,esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂fatty amides, C₇ to C₂₂ ethers, C₇ to C₁₁ fatty alcohols, C₇ to C₁₁fatty acids, esters of C₇ to C₁₁ fatty acids, C₇ to C₁₁ fatty aldehydes,and mixtures thereof. In some embodiments, the second solvent may becarboxylic acids. In some embodiments, the second solvent may be anorganic solvent such as oleyl alcohol, phenyl alcohol, Docosanol(behenyl alcohol), cetyl alcohol, lauryl alcohol (also referred to as1-dodecanol), myristyl alcohol, stearyl alcohol, oleic acid, lauricacid, myristic acid, stearic acid, octanoic acid, decanoic acid,undecanoic acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol,2-undecanol, 1-nonanal, 1-undecanol, undecanal, isododecanol, lauricaldehyde, 2-methylundecanal, oleamide, linoleamide, palmitamide,stearylamide, 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2-octyl-1-dodecanol,or mixtures thereof. Other examples include, but are not limited tophosphates, phosphines, phosphinates, amides, alkylphenols, salicylates,and parabens.

In some embodiments, the extractant may be a mixture of biocompatibleand non-biocompatible extractants. Examples of mixtures of biocompatibleand non-biocompatible extractants include, but are not limited to,isododecane and 2-ethyl-1-hexanol, isododecane and butyl octanol,isododecane and nonanol, isododecane and 1-undecanol, isododecane, and2-undecanol, isododecane, and 1-nonanal, isododecane and decanol,isododecane and dodecanol, oleyl alcohol and nonanol, oleyl alcohol and1-undecanol, oleyl alcohol and 2-undecanol, oleyl alcohol and 1-nonanal,oleyl alcohol and decanol, and oleyl alcohol and dodecanol. Additionalexamples of biocompatible and non-biocompatible extractants aredescribed in U.S. Patent Application Publication No. 2009/0305370 andU.S. Patent Application Publication No. 2011/0097773; the entirecontents of each herein incorporated by reference. In some embodiments,biocompatible extractants may have high atmospheric boiling points. Forexample, biocompatible extractants may have atmospheric boiling pointsgreater than the atmospheric boiling point of water.

In some embodiments, a hydrophilic solute may be added to fermentationbroth that is contacted with an extractant. The presence of ahydrophilic solute in the aqueous broth phase may improve phaseseparation and may increase the fraction of product alcohol thatpartitions into the organic extractant phase. Examples of a hydrophilicsolute may include, but are not limited to, polyhydroxylated,polycarboxylic, polyol compounds and dissociating ionic salts. Sugarssuch as glucose, fructose, sucrose, maltose, and oligosaccharides mayserve as a hydrophilic solute. Other polyhydroxylated compounds mayinclude glycerol, ethylene glycol, propanediol, polyglycerol, andhydroxylated fullerene. Polycarboxylic compounds may include citricacid, tartaric acid, maleic acid, succinic acid, polyacrylic acid, andsodium, potassium, ammonium salts thereof. Ionic salts that may be usedas a hydrophilic solute in fermentation broth comprise cations thatinclude sodium, potassium, ammonium, magnesium, calcium, zinc, andanions that include sulfate, phosphate, chloride, nitrate. The level ofhydrophilic solute in fermentation broth may be selected by one skilledin the art to maximize the transfer of product alcohol out of thefermentation broth phase and into a contacting organic extractant phasewhile not negatively impacting the growth and/or productivity of theproduct alcohol-producing microorganisms. High levels of hydrophilicsolute may impose osmotic stress and/or toxicity to microorganisms infermentation broth. One skilled in the art may use any number of knownmethods to determine an optimal level of hydrophilic solute to minimizethe effects of osmotic stress and/or toxicity on microorganisms.

In some embodiments, the hydrophobic solute may be contacted with theextractant after the extractant is contacted with and separated from thefermentation broth as will be described in further detail in the sectioncaptioned “discussion of sample solvent mixture preparation andextraction” below. Embodiment such as these may be considered two stepprocesses as two different extractions are performed.

In some embodiments, the extractant may comprise an aromatic compound.In some embodiments, the extractant may comprise alkyl substitutedbenzenes including, but not limited to, cumene, para-cymene,meta-cumene, meta-diisopropylbenzene, para-diisopropylbenzene,triisopropylbenzene, tri-sec-butyl-benzene, triethylbenzene, ethyl butylbenzene, tert-butylstyrene. An advantage of using an alkyl substitutedbenzene is the comparatively higher butanol affinity relative to mostother hydrocarbons. In addition, isopropyl, or sec-butyl or isobutylsubstituted benzenes may offer a particular advantage in butanolaffinity over other substituted benzenes. Another advantage is the lowerviscosity, higher interfacial tension, and lower density and higherthermal and chemical stability that aids in the phase separability andlong term reuse.

In embodiments in accordance with the present disclosure, a solventmixture is used to extract alcohol, such as butanol or other fusels froman aqueous solution, such as a fermentation broth. For example, asolvent mixture comprises one or more solvents, such as a first andsecond solvent. The first and second solvents, as well as any additionalsolvents, can be selected to tailor the solvent mixture's properties,although the solvents included in the solvent mixture do not behaveideally. In examples, characteristics such as hydrophobicity, moisturecontent, alcohol affinity, toxicity to a microorganism are consideredwhen selecting which solvents to combine. In implementations, a solventmixture may exhibit properties that are not indicated by the propertiesof the individual solvents (e.g., hydrophobicity, moisture content,alcohol affinity, toxicity) and the mole fraction of the individualsolvents in the solvent mixture. For example, combining a first solventwith a high hydrophobicity with a second solvent that is, in comparisonto the first solvent, less hydrophobic can result in a solvent mixturethat exhibits synergistic alcohol extraction capability beyond thatexpected for the solvent mixture based on the first solvent's propertiesand mole fraction in the solvent mixture and the second solvent'sproperties and its (the second solvent's) mole fraction in the solventmixture. For example, a solvent mixture of corn oil fatty acid (COFA)and isododecane exhibits lower equilibrium moisture content than thatexpected based on the properties of COFA and isododecane on anindividual basis. In embodiments, such as this the aqueous solution thatincludes the alcohol is contacted with the solvent mixture to extractthe alcohol into the solvent mixture.

Distillation of Dry Solvents

An extractant containing butanol (e.g., isobutanol) and water can bestripped to form an extractant that is lean in butanol. In this ternarysystem, there may not exist a method to selectively strip butanol overwater because the two components form what is known as a minimum boilingazeotrope. Therefore, the minimum energy associated with stripping theextractant will include the latent heat of vaporizing the butanol alongwith the latent heat of co-vaporizing water vapor. Because the latentheat of water vapor is greater than (e.g., almost 4 times as much) thelatent heat of butanol on a mass basis, water co-vaporization can resultin an increased energy demand to remove the butanol from the extractant.The overhead vapor generated in stripping the extractant may thereforecontain a mixture of butanol and water and may require condensation,decantation and further distillation to isolate a purified butanolproduct that is suitable for biofuel applications. In the case of a dryextractant, the water content is significantly reduced and so is theco-vaporization energy contribution to the total stripping energy neededto remove butanol. Furthermore, the overhead vapor generated fromstripping butanol from a dry extractant may upon condensation form abutanol product with a water content that is within some specified rangefor biofuel application and in that instance, no further distillation ofthe stream may be required.

In some embodiments, an extractant containing butanol may be phaseseparated from fermentation broth and distilled in a column operatingunder vacuum. This distillation may operate with reflux in order tomaintain a distillate of high purity butanol that contains very littleextractant. The bottoms may comprise a portion of the butanol containedin the distillation feed such that the reboiling temperature undervacuum is suitable for delivering heat indirectly from available steam.Distillation may be carried out with a partial condenser where onlyreflux liquid is condensed, and a vapor distillate of substantiallybutanol composition may be directed into the bottom of a rectificationcolumn that is simultaneously fed a butanol stream decanted fromcondensed beer column overhead vapor. An advantage of this type ofdistillation is that the need for a reboiler to purify the decantedbutanol stream is eliminated by heat integrating the vapor generatedfrom stripping butanol out of the extractant.

Conversion of Isobutanol to Isododecane

In some embodiments, a stream of high purity butanol (e.g., isobutanol)produced can be utilized to produce a derivative of isobutanol. Thederivative of isobutanol can, for example, be tri-isobutylene orisododecane. The stream of high purity butanol can be taken afterdistillation into a vessel. In the vessel, the isobutanol can becatalytically converted to tri-isobutylene and/or isododecane.

Isobutanol can be converted to triisobutylene and/or isododecane asillustrated in FIG. 8. FIG. 8 depicts a typical process configurationfor converting isobutanol to a higher alkane comprising predominantly2,2,4,6,6-pentamethylheptane. Isobutanol is preheated and fed via stream1 to a reaction vessel that contains 2.5% para toluene sulfonic acidoperating at 160° C. and 50 psig. A vapor stream is generated and ispassed up through a rectification column before being partiallycondensed and decanted into two liquid phases at 35° C. and 45 psig. Theupper phase is organic comprising mostly isobutylene and is returned tothe top of the column as reflux while the lower aqueous phase is removedas stream 2. Uncondensed vapors are let down in pressure across a valveand reheated by a steam exchanger to form isobutylene vapor stream 3that is fed into a tubular oligomerization reactor packed withAmberlyst-15 catalyst in the form of 0.5 mm beads operating at 100° C.and near atmospheric pressure. At a weight hourly space velocity of 1 gisobutylene per g catalyst per hour, 8.6% of the isobutylene isconverted to diisobutylene, 81.6% to triisobutylene and 5.8% totetraisobutylene, while 4% remains unconverted. The reactor effluentstream 4 is flashed in a drum to safely vent off the unreactedisobutylene and the mixed isomers of isobutylene oligomers is pumped viastream 5 to a trickle bed hydrogenation reactor along with an excessfeed of hydrogen gas stream 6 sourced from cylinder storage. Theconversion of olefins is quantitative and the hydrogenation reactoreffluent stream 7 is flashed in a drum to safely vent off unreactedhydrogen gas and produce a liquid product steam 8 comprisingpredominantly 2,2,4,6,6-pentamethylheptane. Steps for the conversion ofisobutanol to triisobutylene and isododecane(2,2,4,6,6-pentamethylheptane) are known in the art, see, for e.g.,Alcantara et al., Reactive Funct. Polymers 45:19-27 (2000); Ludwig etal., J. Catalysis 284:148-56 (2011); and U.S. Pat. No. 5,625,109.

Recombinant Microorganisms

While not wishing to be bound by theory, it is believed that theprocesses described herein are useful in conjunction with any alcoholproducing microorganism, particularly recombinant microorganisms whichproduce alcohol.

Recombinant microorganisms which produce alcohol are also known in theart (e.g., Ohta et al., Appl. Environ. Microbiol. 57:893-900 (1991);Underwood et al., Appl. Envrion. Microbiol. 68:1071-81 (2002); Shen andLiao, Metab. Eng. 10:312-20 (2008); Hahnai et al., Appl. Environ.73:7814-8 (2007); U.S. Pat. No. 5,514,583; U.S. Pat. No. 5,712,133;International Publication No. WO 1995/028476; Feldmann et al., Appl.Microbiol. Biotechnol. 38:354-61 (1992); Zhang et al., Science 267:240-3(1995); U.S. Patent Publication No. 2007/0031918A1; U.S. Pat. No.7,223,575; U.S. Pat. No. 7,741,119; U.S. Patent Publication No.2009/0203099A1; U.S. Patent Publication No. 2009/0246846A1; andInternational Publication No. WO 2010/075241, which are hereinincorporated by reference).

For example, the metabolic pathways of microorganisms may be geneticallymodified to produce butanol. These pathways may also be modified toreduce or eliminate undesired metabolites, and thereby improve yield ofthe product alcohol. The production of butanol by a microorganism isdisclosed, for example, in U.S. Pat. Nos. 7,851,188; 7,993,889;8,178,328, 8,206,970; U.S. Patent Application Publication Nos.2007/0292927; 2008/0182308; 2008/0274525; 2009/0305363; 2009/0305370;2011/0250610; 2011/0313206; 2011/0111472; 2012/0258873; and U.S. patentapplication Ser. No. 13/428,585, the entire contents of each are hereinincorporated by reference. In some embodiments, microorganisms comprisea butanol biosynthetic pathway or a biosynthetic pathway for a butanolisomer such as 1-butanol, 2-butanol, or isobutanol. In some embodiments,the biosynthetic pathway converts pyruvate to a fermentative product. Insome embodiments, the biosynthetic pathway converts pyruvate as well asamino acids to a fermentative product. In some embodiments, at leastone, at least two, at least three, or at least four polypeptidescatalyzing substrate to product conversions of a pathway are encoded byheterologous polynucleotides in the microorganism. In some embodiments,all polypeptides catalyzing substrate to product conversions of apathway are encoded by heterologous polynucleotides in themicroorganism.

In some embodiments, the microorganism may be bacteria, cyanobacteria,filamentous fungi, or yeasts. Suitable microorganisms capable ofproducing product alcohol (e.g., butanol) via a biosynthetic pathwayinclude a member of the genera Clostridium, Zymomonas, Escherichia,Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium,Schizosaccharomyces, Kluveromyces, Yarrowia, Pichia, Zygosaccharomyces,Debaryomyces, Candida, Brettanomyces, Pachysolen, Hansenula,Issatchenkia, Trichosporon, Yamadazyma, or Saccharomyces. In oneembodiment, recombinant microorganisms may be selected from the groupconsisting of Escherichia coli, Alcaligenes eutrophus, Bacilluslichenifonnis, Paenibacillus macerans, Rhodocuccus erythropolis,Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis,Candida sonorensis, Candida methanosorbosa, Kluyveromyces lactis,Kluyveromyces marxianus, Kluveromyces thermotolerans, Issatchenkiaorientalis, Debaryomyces hansenii, and Saccharomyces cerevisiae. In oneembodiment, the genetically modified microorganism is yeast. In oneembodiment, the genetically modified microorganism is acrabtree-positive yeast selected from Saccharomyces, Zygosaccharomyces,Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and somespecies of Candida. Species of crabtree-positive yeast include, but arenot limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri,Schizosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces mikitae,Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces castelli,Zygosaccharomyces rouxii, Zygosaccharomyces bailli, and Candidaglabrata.

In some embodiments, the host cell is Saccharomyces cerevisiae.Saccharomyces cerevisiae are known in the art and are available from avariety of sources including, but not limited to, American Type CultureCollection (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS)Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions,North American Bioproducts, Martrex, and Lallemand. S. cerevisiaeinclude, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red®yeast, Ferm Pro™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige BatchTurbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert StrandDistillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast,Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

In some embodiments, the microorganism may be immobilized orencapsulated. For example, the microorganism may be immobilized orencapsulated using alginate, calcium alginate, or polyacrylamide gels,or through the induction of biofilm formation onto a variety of highsurface area support matrices such as diatomite, celite, diatomaceousearth, silica gels, plastics, or resins. In some embodiments, ISPR maybe used in combination with immobilized or encapsulated microorganisms.This combination may improve productivity such as specific volumetricproductivity, metabolic rate, product alcohol yields, tolerance toproduct alcohol. In addition, immobilization and encapsulation mayminimize the effects of the process conditions such as shearing on themicroorganisms.

Biosynthetic pathways for the production of isobutanol that may be usedinclude those as described by Donaldson et al. in U.S. Pat. No.7,851,188; U.S. Pat. No. 7,993,388; and International Publication No. WO2007/050671, which are incorporated herein by reference. In oneembodiment, the isobutanol biosynthetic pathway comprises the followingsubstrate to product conversions:

a) pyruvate to acetolactate, which may be catalyzed, for example, byacetolactate synthase;

b) the acetolactate from step a) to 2,3-dihydroxyisovalerate, which maybe catalyzed, for example, by acetohydroxy acid reductoisomerase;

c) the 2,3-dihydroxyisovalerate from step b) to α-ketoisovalerate, whichmay be catalyzed, for example, by acetohydroxy acid dehydratase;

d) the α-ketoisovalerate from step c) to isobutyraldehyde, which may becatalyzed, for example, by a branched-chain α-keto acid decarboxylase;and,

e) the isobutyraldehyde from step d) to isobutanol, which may becatalyzed, for example, by a branched-chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thefollowing substrate to product conversions:

a) pyruvate to acetolactate, which may be catalyzed, for example, byacetolactate synthase;

b) the acetolactate from step a) to 2,3-dihydroxyisovalerate, which maybe catalyzed, for example, by ketol-acid reductoisomerase;

c) the 2,3-dihydroxyisovalerate from step b) to α-ketoisovalerate, whichmay be catalyzed, for example, by dihydroxyacid dehydratase;

d) the α-ketoisovalerate from step c) to valine, which may be catalyzed,for example, by transaminase or valine dehydrogenase;

e) the valine from step d) to isobutylamine, which may be catalyzed, forexample, by valine decarboxylase;

f) the isobutylamine from step e) to isobutyraldehyde, which may becatalyzed by, for example, omega transaminase; and,

g) the isobutyraldehyde from step f) to isobutanol, which may becatalyzed, for example, by a branched-chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thefollowing substrate to product conversions:

a) pyruvate to acetolactate, which may be catalyzed, for example, byacetolactate synthase;

b) the acetolactate from step a) to 2,3-dihydroxyisovalerate, which maybe catalyzed, for example, by acetohydroxy acid reductoisomerase;

c) the 2,3-dihydroxyisovalerate from step b) to α-ketoisovalerate, whichmay be catalyzed, for example, by acetohydroxy acid dehydratase;

d) the α-ketoisovalerate from step c) to isobutyryl-CoA, which may becatalyzed, for example, by branched-chain keto acid dehydrogenase;

e) the isobutyryl-CoA from step d) to isobutyraldehyde, which may becatalyzed, for example, by acylating aldehyde dehydrogenase; and,

f) the isobutyraldehyde from step e) to isobutanol, which may becatalyzed, for example, by a branched-chain alcohol dehydrogenase.

Biosynthetic pathways for the production of 1-butanol that may be usedinclude those described in U.S. Patent Application Publication No.2008/0182308 and WO2007/041269, which are incorporated herein byreference. In one embodiment, the 1-butanol biosynthetic pathwaycomprises the following substrate to product conversions:

a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example,by acetyl-CoA acetyltransferase;

b) the acetoacetyl-CoA from step a) to 3-hydroxybutyryl-CoA, which maybe catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;

c) the 3-hydroxybutyryl-CoA from step b) to crotonyl-CoA, which may becatalyzed, for example, by crotonase;

d) the crotonyl-CoA from step c) to butyryl-CoA, which may be catalyzed,for example, by butyryl-CoA dehydrogenase;

e) the butyryl-CoA from step d) to butyraldehyde, which may becatalyzed, for example, by butyraldehyde dehydrogenase; and,

f) the butyraldehyde from step e) to 1-butanol, which may be catalyzed,for example, by butanol dehydrogenase.

Biosynthetic pathways for the production of 2-butanol that may be usedinclude those described by Donaldson et al. in U.S. Pat. No. 8,206,970;U.S. Patent Application Publication Nos. 2007/0292927 and 2009/0155870;International Publication Nos. WO 2007/130518 and WO 2007/130521, all ofwhich are incorporated herein by reference. In one embodiment, the2-butanol biosynthetic pathway comprises the following substrate toproduct conversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example,by acetolactate synthase;

b) the alpha-acetolactate from step a) to acetoin, which may becatalyzed, for example, by acetolactate decarboxylase;

c) the acetoin from step b) to 3-amino-2-butanol, which may becatalyzed, for example, acetonin aminase;

d) the 3-amino-2-butanol from step c) to 3-amino-2-butanol phosphate,which may be catalyzed, for example, by aminobutanol kinase;

e) the 3-amino-2-butanol phosphate from step d) to 2-butanone, which maybe catalyzed, for example, by aminobutanol phosphate phosphorylase; and,

f) the 2-butanone from step e) to 2-butanol, which may be catalyzed, forexample, by butanol dehydrogenase.

In another embodiment, the 2-butanol biosynthetic pathway comprises thefollowing substrate to product conversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example,by acetolactate synthase;

b) the alpha-acetolactate from step a) to acetoin, which may becatalyzed, for example, by acetolactate decarboxylase;

c) the acetoin to 2,3-butanediol from step b), which may be catalyzed,for example, by butanediol dehydrogenase;

d) the 2,3-butanediol from step c) to 2-butanone, which may becatalyzed, for example, by dial dehydratase; and,

e) the 2-butanone from step d) to 2-butanol, which may be catalyzed, forexample, by butanol dehydrogenase.

Biosynthetic pathways for the production of 2-butanone that may be usedinclude those described in U.S. Pat. No. 8,206,970 and U.S. PatentApplication Publication Nos. 2007/0292927 and 2009/0155870, which areincorporated herein by reference. In one embodiment, the 2-butanonebiosynthetic pathway comprises the following substrate to productconversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example,by acetolactate synthase;

b) the alpha-acetolactate from step a) to acetoin, which may becatalyzed, for example, by acetolactate decarboxylase;

c) the acetoin from step b) to 3-amino-2-butanol, which may becatalyzed, for example, acetonin aminase;

d) the 3-amino-2-butanol from step c) to 3-amino-2-butanol phosphate,which may be catalyzed, for example, by aminobutanol kinase; and,

e) the 3-amino-2-butanol phosphate from step d) to 2-butanone, which maybe catalyzed, for example, by aminobutanol phosphate phosphorylase.

In another embodiment, the 2-butanone biosynthetic pathway comprises thefollowing substrate to product conversions:

a) pyruvate to alpha-acetolactate, which may be catalyzed, for example,by acetolactate synthase;

b) the alpha-acetolactate from step a) to acetoin which may becatalyzed, for example, by acetolactate decarboxylase;

c) the acetoin from step b) to 2,3-butanediol, which may be catalyzed,for example, by butanediol dehydrogenase;

d) the 2,3-butanediol from step c) to 2-butanone, which may becatalyzed, for example, by diol dehydratase.

The terms “acetohydroxyacid synthase,” “acetolactate synthase,” and“acetolactate synthetase” (abbreviated “ALS”) are used interchangeablyherein to refer to an enzyme that catalyzes the conversion of pyruvateto acetolactate and CO₂. Example acetolactate synthases are known by theEC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego).These enzymes are available from a number of sources, including, but notlimited to, Bacillus subtilis (GenBank Nos: CAB07802.1, Z99122, NCBI(National Center for Biotechnology Information) amino acid sequence,NCBI nucleotide sequence, respectively), CAB15618, Klebsiella pneumoniae(GenBank Nos: AAA25079, M73842), and Lactococcus lactis (GenBank Nos:AAA25161, L16975)

The term “ketol-acid reductoisomerase” (“KARI”), “acetohydroxy acidisomeroreductase,” and “acetohydroxy acid reductoisomerase” will be usedinterchangeably and refer to enzymes capable of catalyzing the reactionof (S)-acetolactate to 2,3-dihydroxyisovalerate. Example KARI enzymesmay be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992,Academic Press, San Diego), and are available from a vast array ofmicroorganisms, including, but not limited to, Escherichia coli (GenBankNos: NP_418222, NC_000913), Saccharomyces cerevisiae (GenBank Nos:NP_013459, NC_001144), Methanococcus maripaludis (GenBank Nos: CAF30210,BX957220), Bacillus subtilis (GenBank Nos: CAB14789, Z99118), andAnaerostipes caccae. Ketol-acid reductoisomerase (KARI) enzymes aredescribed in U.S. Pat. Nos. 7,910,342 and 8,129,162; U.S. PatentApplication Publication Nos. 2008/0261230, 2009/0163376, 2010/0197519,PCT Application Publication No. WO/2011/041415, PCT ApplicationPublication No. WO2012/129555; and U.S. Provisional Application No.61/705,977, filed on Sep. 26, 2012, all of which are incorporated hereinby reference. Examples of KARIs disclosed therein are those fromLactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, andPseudomonas fluorescens PF5 mutants. In some embodiments, the KARIutilizes NADH. In some embodiments, the KARI utilizes NADPH. In someembodiments, the KARI utilizes NADH or NADPH.

The term “acetohydroxy acid dehydratase” and “dihydroxyacid dehydratase”(“DHAD”) refers to an enzyme that catalyzes the conversion of2,3-dihydroxyisovalerate to α-ketoisovalerate. Example acetohydroxy aciddehydratases are known by the EC number 4.2.1.9. Such enzymes areavailable from a vast array of microorganisms, including, but notlimited to, E. coli (GenBank Nos: YP_026248, NC000913), Saccharomycescerevisiae (GenBank Nos: NP_012550, NC_001142), M. maripaludis (GenBankNos: CAF29874, BX957219), B. subtilis (GenBank Nos: CAB14105, Z99115),L. lactis, and N. crassa. U.S. Patent Application Publication No.2010/0081154, U.S. Pat. No. 7,851,188, and U.S. Pat. No. 8,241,878,which are incorporated herein by reference in their entireties, describedihydroxyacid dehydratases (DHADs), including a DHAD from Streptococcusmutans and variants thereof.

The term “branched-chain α-keto acid decarboxylase,” “α-ketoaciddecarboxylase,” “α-ketoisovalerate decarboxylase,” or “2-ketoisovaleratedecarboxylase” (“KIVD”) refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to isobutyraldehyde and CO₂. Examplebranched-chain α-keto acid decarboxylases are known by the EC number4.1.1.72 and are available from a number of sources, including, but notlimited to, Lactococcus lactis (GenBank Nos: AAS49166, AY548760;CAG34226, AJ746364), Salmonella typhimurium (GenBank Nos: NP_461346,NC_003197), Clostridium acetobutylicum (GenBank Nos: NP_149189,NC_001988), M. caseolyticus, and L. grayi.

The term “branched-chain alcohol dehydrogenase” (“ADH”) refers to anenzyme that catalyzes the conversion of isobutyraldehyde to isobutanol.Example branched-chain alcohol dehydrogenases are known by the EC number1.1.1.265, but may also be classified under other alcohol dehydrogenases(specifically, EC 1.1.1.1 or 1.1.1.2). Alcohol dehydrogenases may beNADPH dependent or NADH dependent. Such enzymes are available from anumber of sources, including, but not limited to, S. cerevisiae (GenBankNos: NP_010656, NC_001136, NP_014051, NC_001145), E. coli (GenBank Nos:NP_417484, NC_000913), C. acetobutylicum (GenBank Nos: NP_349892,NC_003030; NP_349891, NC_003030). U.S. Patent Application PublicationNo. 2009/0269823 describes SadB, an alcohol dehydrogenase (ADH) fromAchromobacter xylosoxidans. Alcohol dehydrogenases can also includehorse liver ADH and Beijerinkia indica ADH, as described by U.S. PatentApplication Publication No. 2011/0269199, which is incorporated hereinby reference in its entirety.

The term “butanol dehydrogenase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofisobutyraldehyde to isobutanol or the conversion of 2-butanone and2-butanol. Butanol dehydrogenases are a subset of a broad family ofalcohol dehydrogenases. Butanol dehydrogenase may be NAD- orNADP-dependent. The NAD-dependent enzymes are known as EC 1.1.1.1 andare available, for example, from Rhodococcus ruber (GenBank Nos:CAD36475, AJ491307). The NADP dependent enzymes are known as EC 1.1.1.2and are available, for example, from Pyrococcus furiosus (GenBank Nos:AAC25556, AF013169). Additionally, a butanol dehydrogenase is availablefrom Escherichia coli (GenBank Nos: NP_417484, NC_000913) and acyclohexanol dehydrogenase is available from Acinetobacter sp. (GenBankNos: AAG10026, AF282240). The term “butanol dehydrogenase” also refersto an enzyme that catalyzes the conversion of butyraldehyde to1-butanol, using either NADH or NADPH as cofactor. Butanoldehydrogenases are available from, for example, C. acetobutylicum(GenBank NOs: NP_149325, NC_001988; note: this enzyme possesses bothaldehyde and alcohol dehydrogenase activity); NP_349891, NC_003030; andNP_349892, NC_003030) and E. coli (GenBank NOs: NP_417-484, NC_000913).

The term “branched-chain keto acid dehydrogenase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyryl-CoA(isobutyryl-coenzyme A), typically using NAD⁺ (nicotinamide adeninedinucleotide) as an electron acceptor. Example branched-chain keto aciddehydrogenases are known by the EC number 1.2.4.4. Such branched-chainketo acid dehydrogenases are comprised of four subunits and sequencesfrom all subunits are available from a vast array of microorganisms,including, but not limited to, B. subtilis (GenBank Nos: CAB14336,Z99116; CAB14335, Z99116; CAB14334, Z99116; and CAB14337, Z99116) andPseudomonas putida (GenBank Nos: AAA65614, M57613; AAA65615, M57613;AAA65617, M57613; and AAA65618, M57613).

The term “acylating aldehyde dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyryl-CoA to isobutyraldehyde,typically using either NADH or NADPH as an electron donor. Exampleacylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10and 1.2.1.57. Such enzymes are available from multiple sources,including, but not limited to, Clostridium beijerinckii (GenBank Nos:AAD31841, AF157306), C. acetobutylicum (GenBank Nos: NP_149325,NC_001988; NP_149199, NC_001988), P. putida (GenBank Nos: AAA89106,U13232), and Thermus thermophilus (GenBank Nos: YP_145486, NC_006461).

The term “transaminase” refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to L-valine, using either alanine orglutamate as an amine donor. Example transaminases are known by the ECnumbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from a numberof sources. Examples of sources for alanine-dependent enzymes include,but are not limited to, E. coli (GenBank Nos: YP_026231, NC_000913) andBacillus licheniformis (GenBank Nos: YP_093743, NC_006322). Examples ofsources for glutamate-dependent enzymes include, but are not limited to,E. coli (GenBank Nos: YP_026247, NC_000913), Saccharomyces cerevisiae(GenBank Nos: NP_012682, NC_001142) and Methanobacteriumthermoautotrophicum (GenBank Nos: NP_276546, NC_000916).

The term “valine dehydrogenase” refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to L-valine, typically using NAD(P)H asan electron donor and ammonia as an amine donor. Example valinedehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and suchenzymes are available from a number of sources, including, but notlimited to, Streptomyces coelicolor (GenBank Nos: NP_628270, NC_003888)and B. subtilis (GenBank Nos: CAB14339, Z99116).

The term “valine decarboxylase” refers to an enzyme that catalyzes theconversion of L-valine to isobutylamine and CO₂. Example valinedecarboxylases are known by the EC number 4.1.1.14. Such enzymes arefound in Streptomyces, such as for example, Streptomyces viridifaciens(GenBank Nos: AAN10242, AY116644).

The term “omega transaminase” refers to an enzyme that catalyzes theconversion of isobutylamine to isobutyraldehyde using a suitable aminoacid as an amine donor. Example omega transaminases are known by the ECnumber 2.6.1.18 and are available from a number of sources, including,but not limited to, Alcaligenes denitrificans (AAP92672, AY330220),Ralstonia eutropha (GenBank Nos: YP_294474, NC_007347), Shewanellaoneidensis (GenBank Nos: NP_719046, NC_004347), and P. putida (GenBankNos: AAN66223, AE016776).

The term “acetyl-CoA acetyltransferase” refers to an enzyme thatcatalyzes the conversion of two molecules of acetyl-CoA toacetoacetyl-CoA and coenzyme A (CoA). Example acetyl-CoAacetyltransferases are acetyl-CoA acetyltransferases with substratepreferences (reaction in the forward direction) for a short chainacyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [EnzymeNomenclature 1992, Academic Press, San Diego]; although, enzymes with abroader substrate range (E.C. 2.3.1.16) will be functional as well.Acetyl-CoA acetyltransferases are available from a number of sources,for example, Escherichia coli (GenBank Nos: NP_416728, NC_000913; NCBI(National Center for Biotechnology Information) amino acid sequence,NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos:NP_349476.1, NC_003030; NP_149242, NC_001988, Bacillus subtilis (GenBankNos: NP_390297, NC_000964), and Saccharomyces cerevisiae (GenBank Nos:NP_015297, NC_001148).

The term “3-hydroxybutyryl-CoA dehydrogenase” refers to an enzyme thatcatalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA.3-Example hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamideadenine dinucleotide (NADH)-dependent, with a substrate preference for(S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA. Examples may beclassified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively.Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be reducednicotinamide adenine dinucleotide phosphate (NADPH)-dependent, with asubstrate preference for (S)-3-hydroxybutyryl-CoA or(R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C.1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases areavailable from a number of sources, for example, C. acetobutylicum(GenBank NOs: NP_349314, NC_003030), B. subtilis (GenBank NOs: AAB09614,U29084), Ralstonia eutropha (GenBank NOs: YP_294481, NC_007347), andAlcaligenes eutrophus (GenBank NOs: AAA21973, J04987).

The term “crotonase” refers to an enzyme that catalyzes the conversionof 3-hydroxybutyryl-CoA to crotonyl-CoA and H₂O. Example crotonases mayhave a substrate preference for (S)-3-hydroxybutyryl-CoA or(R)-3-hydroxybutyryl-CoA and may be classified as E.C. 4.2.1.17 and E.C.4.2.1.55, respectively. Crotonases are available from a number ofsources, for example, E. coli (GenBank NOs: NP_415911, NC_000913), C.acetobutylicum (GenBank NOs: NP_349318, NC_003030), B. subtilis (GenBankNOs: CAB13705, Z99113), and Aeromonas caviae (GenBank NOs: BAA21816,D88825).

The term “butyryl-CoA dehydrogenase” refers to an enzyme that catalyzesthe conversion of crotonyl-CoA to butyryl-CoA. Example butyryl-CoAdehydrogenases may be NADH-dependent, NADPH-dependent, orflavin-dependent and may be classified as E.C. 1.3.1.44, E.C. 1.3.1.38,and E.C. 1.3.99.2, respectively. Butyryl-CoA dehydrogenases areavailable from a number of sources, for example, C. acetobutylicum(GenBank NOs: NP_347102, NC_(—) 003030), Euglena gracilis (GenBank NOs:Q5EU90, AY741582), Streptomyces collinus (GenBank NOs: AAA92890,U37135), and Streptomyces coelicolor (GenBank NOs: CAA22721, AL939127).

The term “butyraldehyde dehydrogenase” refers to an enzyme thatcatalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH orNADPH as cofactor. Butyraldehyde dehydrogenases with a preference forNADH are known as E.C. 1.2.1.57 and are available from, for example,Clostridium beijerinckii (GenBank NOs: AAD31841, AF157306) and C.acetobutylicum (GenBank NOs: NP_149325, NC_001988).

The term “isobutyryl-CoA mutase” refers to an enzyme that catalyzes theconversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzymeB₁₂ as cofactor. Example isobutyryl-CoA mutases are known by the ECnumber 5.4.99.13. These enzymes are found in a number of Streptomyces,including, but not limited to, Streptomyces cinnamonensis (GenBank Nos:AAC08713, U67612; CAB59633, AJ246005), S. coelicolor (GenBank Nos:CAB70645, AL939123; CAB92663, AL939121), and Streptomyces avermitilis(GenBank Nos: NP_824008, NC_003155; NP_824637, NC_003155).

The term “acetolactate decarboxylase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofalpha-acetolactate to acetoin. Example acetolactate decarboxylases areknown as EC 4.1.1.5 and are available, for example, from Bacillussubtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBankNos: AAA25054, L04507) and Klebsiella pneumoniae (GenBank Nos: AAU43774,AY722056).

The term “acetoin aminase” or “acetoin transaminase” refers to apolypeptide (or polypeptides) having an enzyme activity that catalyzesthe conversion of acetoin to 3-amino-2-butanol. Acetoin aminase mayutilize the cofactor pyridoxal 5′-phosphate or NADH (reducednicotinamide adenine dinucleotide) or NADPH (reduced nicotinamideadenine dinucleotide phosphate). The resulting product may have (R) or(S) stereochemistry at the 3-position. The pyridoxal phosphate-dependentenzyme may use an amino acid such as alanine or glutamate as the aminodonor. The NADH- and NADPH-dependent enzymes may use ammonia as a secondsubstrate. A suitable example of an NADH dependent acetoin aminase, alsoknown as amino alcohol dehydrogenase, is described by Ito, et al. (U.S.Pat. No. 6,432,688). An example of a pyridoxal-dependent acetoin aminaseis the amine:pyruvate aminotransferase (also called amine:pyruvatetransaminase) described by Shin and Kim (J. Org. Chem. 67:2848-2853,2002).

The term “acetoin kinase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of acetoin tophosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate)or phosphoenolpyruvate as the phosphate donor in the reaction. Enzymesthat catalyze the analogous reaction on the similar substratedihydroxyacetone, for example, include enzymes known as EC 2.7.1.29(Garcia-Alles, et al., Biochemistry 43:13037-13046, 2004).

The term “acetoin phosphate aminase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofphosphoacetoin to 3-amino-2-butanol O-phosphate. Acetoin phosphateaminase may use the cofactor pyridoxal 5′-phosphate, NADH or NADPH. Theresulting product may have (R) or (S) stereochemistry at the 3-position.The pyridoxal phosphate-dependent enzyme may use an amino acid such asalanine or glutamate. The NADH and NADPH-dependent enzymes may useammonia as a second substrate. Although there are no reports of enzymescatalyzing this reaction on phosphoacetoin, there is a pyridoxalphosphate-dependent enzyme that is proposed to carry out the analogousreaction on the similar substrate serinol phosphate (Yasuta, et al.,Appl. Environ. Microbial. 67:4999-5009, 2001).

The term “aminobutanol phosphate phospholyase,” also called “aminoalcohol O-phosphate lyase,” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of3-amino-2-butanol O-phosphate to 2-butanone. Amino butanol phosphatephospho-lyase may utilize the cofactor pyridoxal 5′-phosphate. There arereports of enzymes that catalyze the analogous reaction on the similarsubstrate 1-amino-2-propanol phosphate (Jones, et al., Biochem J.134:167-182, 1973). U.S. Patent Application Publication No. 2007/0259410describes an aminobutanol phosphate phospho-lyase from the organismErwinia carotovora.

The term “aminobutanol kinase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Amino butanol kinasemay utilize ATP as the phosphate donor. Although there are no reports ofenzymes catalyzing this reaction on 3-amino-2-butanol, there are reportsof enzymes that catalyze the analogous reaction on the similarsubstrates ethanolamine and 1-amino-2-propanol (Jones, et al., supra).U.S. Patent Application Publication No. 2009/0155870 describes, inExample 14, an amino alcohol kinase of Erwinia carotovora subsp.Atroseptica.

The term “butanediol dehydrogenase” also known as “acetoin reductase”refers to a polypeptide (or polypeptides) having an enzyme activity thatcatalyzes the conversion of acetoin to 2,3-butanediol. Butanedialdehydrogenases are a subset of the broad family of alcoholdehydrogenases. Butanediol dehydrogenase enzymes may have specificityfor production of (R)- or (S)-stereochemistry in the alcohol product.(S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and areavailable, for example, from Klebsiella pneumoniae (GenBank Nos:BBA13085, D86412). (R)-specific butanediol dehydrogenases are known asEC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBankNos. NP_830481, NC_004722; AAP07682, AE017000), and Lactococcus lactis(GenBank Nos. AAK04995, AE006323).

The term “butanediol dehydratase,” also known as “dial dehydratase” or“propanediol dehydratase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of2,3-butanediol to 2-butanone. Butanediol dehydratase may utilize thecofactor adenosyl cobalamin (also known as coenzyme Bw or vitamin B₁₂;although vitamin B12 may refer also to other forms of cobalamin that arenot coenzyme B12). Adenosyl cobalamin-dependent enzymes are known as EC4.2.1.28 and are available, for example, from Klebsiella oxytoca(GenBank Nos: AA08099 (alpha subunit), D45071; BAA08100 (beta subunit),D45071; and BBA08101 (gamma subunit), D45071 (Note all three subunitsare required for activity), and Klebsiella pneumonia (GenBank Nos:AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (betasubunit), AF102064, GenBank Nos: AAC98386 (gamma subunit), AF102064).Other suitable dial dehydratases include, but are not limited to,B12-dependent dial dehydratases available from Salmonella typhimurium(GenBank Nos: AAB84102 (large subunit), AF026270; GenBank Nos: AAB84103(medium subunit), AF026270; GenBank Nos: AAB84104 (small subunit),AF026270); and Lactobacillus collinoides (GenBank Nos: CAC82541 (largesubunit), AJ297723; GenBank Nos: CAC82542 (medium subunit); AJ297723;GenBank Nos: CAD01091 (small subunit), AJ297723); and enzymes fromLactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735,Speranza, et al., J. Agric. Food Chem. 45:3476-3480, 1997), andnucleotide sequences that encode the corresponding enzymes. Methods ofdiol dehydratase gene isolation are well known in the art (e.g., U.S.Pat. No. 5,686,276).

The term “pyruvate decarboxylase” refers to an enzyme that catalyzes thedecarboxylation of pyruvic acid to acetaldehyde and carbon dioxide.Pyruvate dehydrogenases are known by the EC number 4.1.1.1. Theseenzymes are found in a number of yeast, including Saccharomycescerevisiae (GenBank Nos: CAA97575, CAA97705, CAA97091).

It will be appreciated that host cells comprising an isobutanolbiosynthetic pathway as provided herein may further comprise one or moreadditional modifications. U.S. Patent Application Publication No.2009/0305363 (incorporated by reference) discloses increased conversionof pyruvate to acetolactate by engineering yeast for expression of acytosol-localized acetolactate synthase and substantial elimination ofpyruvate decarboxylase activity. In some embodiments, the host cellscomprise modifications to reduce glycerol-3-phosphate dehydrogenaseactivity and/or disruption in at least one gene encoding a polypeptidehaving pyruvate decarboxylase activity or a disruption in at least onegene encoding a regulatory element controlling pyruvate decarboxylasegene expression as described in U.S. Patent Application Publication No.2009/0305363 (incorporated herein by reference), modifications to a hostcell that provide for increased carbon flux through an Entner-DoudoroffPathway or reducing equivalents balance as described in U.S. PatentApplication Publication No. 2010/0120105 (incorporated herein byreference). Other modifications include integration of at least onepolynucleotide encoding a polypeptide that catalyzes a step in apyruvate-utilizing biosynthetic pathway.

Other modifications include at least one deletion, mutation, and/orsubstitution in an endogenous polynucleotide encoding a polypeptidehaving acetolactate reductase activity. As used herein, “acetolactatereductase activity” refers to the activity of any polypeptide having theability to catalyze the conversion of acetolactate to DHMB. Suchpolypeptides can be determined by methods well known in the art anddisclosed herein. As used herein, “DHMB” refers to2,3-dihydroxy-2-methyl butyrate. DHMB includes “fast DHMB,” which hasthe 2S, 3S configuration, and “slow DHMB,” which has the 2S, 3Rconfigurate. See Kaneko et al., Phytochemistry 39: 115-120 (1995), whichis herein incorporated by reference in its entirety and refers to fastDHMB as anglyceric acid and slow DHMB as tiglyceric acid. Inembodiments, the polypeptide having acetolactate reductase activity isYMR226C of Saccharomyces cerevisiae or a homolog thereof.

Additional modifications include a deletion, mutation, and/orsubstitution in an endogenous polynucleotide encoding a polypeptidehaving aldehyde dehydrogenase and/or aldehyde oxidase activity. As usedherein, “aldehyde dehydrogenase activity” refers to any polypeptidehaving a biological function of an aldehyde dehydrogenase. Suchpolypeptides include a polypeptide that catalyzes the oxidation(dehydrogenation) of aldehydes. Such polypeptides include a polypeptidethat catalyzes the conversion of isobutyraldehyde to isobutyric acid.Such polypeptides also include a polypeptide that corresponds to EnzymeCommission Numbers EC 1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5. Suchpolypeptides can be determined by methods well known in the art anddisclosed herein. As used herein, “aldehyde oxidase activity” refers toany polypeptide having a biological function of an aldehyde oxidase.Such polypeptides include a polypeptide that catalyzes production ofcarboxylic acids from aldehydes. Such polypeptides include a polypeptidethat catalyzes the conversion of isobutyraldehyde to isobutyric acid.Such polypeptides also include a polypeptide that corresponds to EnzymeCommission Number EC 1.2.3.1. Such polypeptides can be determined bymethods well known in the art and disclosed herein. In some embodiments,the polypeptide having aldehyde dehydrogenase activity is ALD6 fromSaccharomyces cerevisiae or a homolog thereof.

A genetic modification which has the effect of reducing glucoserepression wherein the yeast production host cell is pdc- is describedin U.S. Patent Application Publication No. 2011/0124060, incorporatedherein by reference. In some embodiments, the pyruvate decarboxylasethat is deleted or down-regulated is selected from the group consistingof: PDC1, PDC5, PDC6, and combinations thereof. In some embodiments, thepyruvate decarboxylase is selected from PDC1 pyruvate decarboxylase fromSaccharomyces cerevisiae, PDC5 pyruvate decarboxylase from Saccharomycescerevisiae, PDC6 pyruvate decarboxylase from Saccharomyces cerevisiae,pyruvate decarboxylase from Candida glabrata, PDC1 pyruvatedecarboxylase from Pichia stipites, PDC2 pyruvate decarboxylase fromPichia stipites, pyruvate decarboxylase from Kluveromyces lactis,pyruvate decarboxylase from Yarrowia lipolytica, pyruvate decarboxylasefrom Schizosaccharomyces pombe, and pyruvate decarboxylase fromZygosaccharomyces rouxii. In some embodiments, host cells contain adeletion or down-regulation of a polynucleotide encoding a polypeptidethat catalyzes the conversion of glyceraldehyde-3-phosphate to glycerate1,3, bisphosphate. In some embodiments, the enzyme that catalyzes thisreaction is glyceraldehyde-3-phosphate dehydrogenase.

In contrast, in an isobutanologen (PDC-) strain, PDC is deleted, the PDHpathway remains intact, and isobutanol production pathway enzymes areintroduced. Often, the first enzyme to act in the isobutanol productionpathway is acetolactate synthase (ALS). In isobutanologens, the carbonflux distribution for biomass growth and for the isobutanol pathwayunder aerobic conditions depends on the relative activity of ALS insteadof the PDH enzyme. The physiological behavior of a recombinantisobutanologen is different from an unmodified S. cerevisiae due to theeffect of the deletion of PDC genes and introduction of heterologousisobutanol pathway enzymes. To maximize biomass production in arecombinant isobutanologen in aerobic growth phase, the carbon flux hasto channel through the PDH pathway efficiently to improve biomass yieldand minimize carbon flux to isobutanol pathway leakages. Pathway leakageproducts can include isobutanol and isobutyric acid, which can adverselyaffect biomass growth rate and the final biomass achieved. In theproduction phase, the isobutanol yield and productivity can be adverselyaffected by accumulation of pathway intermediates (e.g., glycerol andisobutyric acid). Thus, the optimal operating regime (growth andproduction) for an ethanologen may not be the optimal operating regimefor an isobutanologen.

WIPO publication number WO 2001/103300 discloses recombinant host cellscomprising (a) at least one heterologous polynucleotide encoding apolypeptide having dihydroxy-acid dehydratase activity; and (b)(i) atleast one deletion, mutation, and/or substitution in an endogenous geneencoding a polypeptide affecting Fe—S cluster biosynthesis; and/or (ii)at least one heterologous polynucleotide encoding a polypeptideaffecting Fe—S cluster biosynthesis. In embodiments, the polypeptideaffecting Fe—S cluster biosynthesis is encoded by AFT1, AFT2, FRA2,GRX3, or CCC1. In embodiments, the polypeptide affecting Fe—S clusterbiosynthesis is constitutive mutant AFT1 L99A, AFT1 L102A, AFT1 C291F,or AFT1 C293F.

Additionally, host cells may comprise heterologous polynucleotidesencoding a polypeptide with phosphoketolase activity and/or aheterologous polynucleotide encoding a polypeptide withphosphotransacetylase activity.

In some embodiments, any particular nucleic acid molecule or polypeptidemay be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to anucleotide sequence or polypeptide sequence described herein. The term“percent identity” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedisclosed in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.)Oxford University: NY (1988); 2.) Biocomputing: Informatics and GenomeProjects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysisof Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.)Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (vonHeinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer(Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Standard recombinant DNA and molecular cloning techniques are well knownin the art and are described by Sambrook, et al. (Sambrook, J., Fritsch,E. F. and Maniatis, T. (Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 1989, here inreferred to as Maniatis) and by Ausubel, et al. (Ausubel, et al.,Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc.and Wiley-Interscience, 1987). Examples of methods to constructmicroorganisms that comprise a butanol biosynthetic pathway aredisclosed, for example, in U.S. Pat. No. 7,851,188, and U.S. PatentApplication Publication Nos. 2007/0092957; 2007/0259410; 2007/0292927;2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; and2009/0305370, the entire contents of each are herein incorporated byreference.

Growth for Production

Recombinant host cells disclosed herein are contacted with suitablecarbon substrates, typically in fermentation media. Additional carbonsubstrates may include, but are not limited to, monosaccharides such asfructose, oligosaccharides such as lactose, maltose, galactose, orsucrose, polysaccharides such as starch or cellulose or mixtures thereofand unpurified mixtures from renewable feedstocks such as cheese wheypermeate, cornsteep liquor, sugar beet molasses, and barley malt. Othercarbon substrates can include ethanol, lactate, succinate, or glycerol.

Additionally the carbon substrate may also be one-carbon substrates suchas carbon dioxide, or methanol for which metabolic conversion into keybiochemical intermediates has been demonstrated. In addition to one andtwo carbon substrates, methylotrophic organisms are also known toutilize a number of other carbon containing compounds such asmethylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeasts are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth Cl Compd., [Int. Symp.], 7^(th) (1993), 415-32, Editors:Murrell, J. Collin, Kelly, Don P.; Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,in some embodiments, the carbon substrates are glucose, fructose, andsucrose, or mixtures of these with C5 sugars such as xylose and/orarabinose for yeasts cells modified to use C5 sugars. Sucrose may bederived from renewable sugar sources such as sugar cane, sugar beets,cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose canbe derived from renewable grain sources through saccharification ofstarch based feedstocks including grains such as corn, wheat, rye,barley, oats, and mixtures thereof. In addition, fermentable sugars canbe derived from renewable cellulosic or lignocellulosic biomass throughprocesses of pretreatment and saccharification, as described, forexample, in U.S. Patent Application Publication No. 2007/0031918 A1,which is herein incorporated by reference. Biomass, when used inreference to carbon substrate, refers to any cellulosic orlignocellulosic material and includes materials comprising cellulose,and optionally further comprising hemicellulose, lignin, starch,oligosaccharides and/or monosaccharides. Biomass can also compriseadditional components, such as protein and/or lipid. Biomass can bederived from a single source, or biomass can comprise a mixture derivedfrom more than one source; for example, biomass may comprise a mixtureof corn cobs and corn stover, or a mixture of grass and leaves. Biomassincludes, but is not limited to, bioenergy crops, agricultural residues,municipal solid waste, industrial solid waste, sludge from papermanufacture, yard waste, wood and forestry waste. Examples of biomassinclude, but are not limited to, corn grain, corn cobs, crop residuessuch as corn husks, corn stover grasses, wheat, wheat straw, barley,barley straw, hay, rice straw, switchgrass, waste paper, sugar canebagasse, sorghum, soy, components obtained from milling of grains,trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers, animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of an enzymatic pathway described herein.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C.to about 40° C. in an appropriate medium. Suitable growth media in thepresent invention are common commercially prepared media such as LuriaBertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM)broth or broth that includes yeast nitrogen base, ammonium sulfate, anddextrose (as the carbon/energy source) or YPD Medium, a blend ofpeptone, yeast extract, and dextrose in optimal proportions for growingmost Saccharomyces cerevisiae strains. Other defined or synthetic growthmedia can also be used, and the appropriate medium for growth of theparticular microorganism will be known by one skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′,3′-monophosphate (cAMP), can also be incorporated into thefermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred for the initial condition. SuitablepH ranges for the fermentation of yeast are typically between about pH3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 isused for the initial condition. Suitable pH ranges for the fermentationof other microorganisms are between about pH 3.0 to about pH 7.5. In oneembodiment, about pH 4.5 to about pH 6.5 is used for the initialcondition.

Fermentations can be performed under aerobic or anaerobic conditions. Inone embodiment, anaerobic or microaerobic conditions are used forfermentation.

Industrial Batch and Continuous Fermentations

Butanol, or other products, can be produced using a batch method offermentation. A classical batch fermentation is a closed system wherethe composition of the medium is set at the beginning of thefermentation and not subject to artificial alterations during thefermentation. A variation on the standard batch system is the fed-batchsystem. Fed-batch fermentation processes are also suitable in thepresent invention and comprise a typical batch system with the exceptionthat the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression isapt to inhibit the metabolism of the cells and where it is desirable tohave limited amounts of substrate in the media. Batch and fed-batchfermentations are common and well known in the art and examples can befound in Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol.,36:227, (1992), herein incorporated by reference.

Butanol, or other products, may also be produced using continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.Methods of modulating nutrients and growth factors for continuousfermentation processes as well as techniques for maximizing the rate ofproduct formation are well known in the art of industrial microbiologyand a variety of methods are detailed by Brock, supra.

It is contemplated that the production of butanol, or other products,can be practiced using batch, fed-batch or continuous processes and thatany known mode of fermentation would be suitable. Additionally, it iscontemplated that cells can be immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for butanolproduction.

Methods for Recovering Butanol using Extractive Fermentation

Bioproduced butanol may be recovered from a fermentation mediumcontaining butanol, water, at least one fermentable carbon source, and amicroorganism that has been genetically modified (that is, geneticallyengineered) to produce butanol via a biosynthetic pathway from at leastone carbon source. The first step in the process is contacting thefermentation medium with a water immiscible organic extractantcomposition comprising a solvent, as described above, to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase. “Contacting” means the fermentation medium and theorganic extractant composition or its solvent component(s) are broughtinto physical contact at any time during the fermentation process. Inone embodiment, the fermentation medium further comprises ethanol, andthe butanol-containing organic phase can contain ethanol.

In certain embodiments where more than one solvent is used for theextraction, the contacting may be performed with the solvents of theextractant composition having been previously combined. For example, thefirst and second solvents may be combined in a vessel such as a mixingtank to form the extractant, which is then added to a vessel containingthe fermentation medium. Alternatively, the contacting may be performedwith the first and second solvents becoming combined during thecontacting. For example, the first and second solvents may be addedseparately to a vessel which contains the fermentation medium. In oneembodiment, contacting the fermentation medium with the organicextractant composition further comprises contacting the fermentationmedium with the first solvent prior to contacting the fermentationmedium and the first solvent with the second solvent. In one embodiment,the contacting with the second solvent occurs in the same vessel as thecontacting with the first solvent. In one embodiment, the contactingwith the second solvent occurs in a different vessel from the contactingwith the first solvent. For example, the first solvent may be contactedwith the fermentation medium in one vessel, and the contents transferredto another vessel in which contacting with the second solvent occurs.

The organic extractant composition may contact the fermentation mediumat the start of the fermentation forming a biphasic fermentation medium.Alternatively, the organic extractant composition may contact thefermentation medium after the microorganism has achieved a desiredamount of growth, which can be determined by measuring the opticaldensity of the culture.

Further, the organic extractant composition may contact the fermentationmedium at a time at which the butanol level in the fermentation mediumreaches a preselected level, for example, before the butanolconcentration reaches a toxic level. The butanol concentration may bemonitored during the fermentation using methods known in the art, suchas by gas chromatography or high performance liquid chromatography.

Fermentation may be run under aerobic conditions for a time sufficientfor the culture to achieve a preselected level of growth, as determinedby optical density measurement. An inducer may then be added to inducethe expression of the butanol biosynthetic pathway in the modifiedmicroorganism, and fermentation conditions are switched to microaerobicor anaerobic conditions to stimulate butanol production, as described indetail in Example 6 of US Patent Application Publication No.2009/0305370 A1. The extractant is added after the switch tomicroaerobic or anaerobic conditions.

Through contacting the fermentation medium with the organic extractant,the butanol product partitions into the organic extractant, decreasingthe concentration in the aqueous phase containing the microorganism,thereby limiting the exposure of the production microorganism to theinhibitory butanol product. The volume of the organic extractant to beused depends on a number of factors, including the volume of thefermentation medium, the size of the fermentor, the partitioncoefficient of the extractant for the butanol product, and thefermentation mode chosen, as described below. The volume of the organicextractant may be about 3% to about 60% of the fermentor working volume.The ratio of the extractant to the fermentation medium is from about1:20 to about 20:1 on a volume:volume basis, for example from about 1:15to about 15:1, or from about 1:12 to about 12:1, or from about 1:10 toabout 10:1, or from about 1:9 to about 9:1, or from about 1:8 to about8:1.

The next step is separating the butanol-containing organic phase fromthe aqueous phase using methods known in the art, including but notlimited to, siphoning, decantation, centrifugation, using a gravitysettler, membrane-assisted phase splitting, and the like. Recovery ofthe butanol from the butanol-containing organic phase can be done usingmethods known in the art, including but not limited to, distillation,adsorption by resins, separation by molecular sieves, pervaporation, andthe like. Specifically, distillation may be used to recover the butanolfrom the butanol-containing organic phase. The extractant or thesolvents may be recycled to the butanol production and/or recoveryprocess.

Gas stripping may be used concurrently with the solvents of the organicextractant composition to remove the butanol product from thefermentation medium. Gas stripping may be done by passing a gas such asair, nitrogen, or carbon dioxide through the fermentation medium,thereby forming a butanol-containing gas phase. The butanol product maybe recovered from the butanol-containing gas phase using methods knownin the art, such as using a chilled water trap to condense the butanol,or scrubbing the gas phase with a solvent.

Any butanol remaining in the fermentation medium after the fermentationrun is completed may be recovered by continued extraction using fresh orrecycled organic extractant. Alternatively, the butanol can be recoveredfrom the fermentation medium using methods known in the art, including,but not limited to distillation, azeotropic distillation, liquid-liquidextraction, adsorption, gas stripping, membrane evaporation,pervaporation, and the like.

The two-phase extractive fermentation method may be carried out in acontinuous mode in a stirred tank fermentor. In this mode, the mixtureof the fermentation medium and the butanol-containing organic extractantcomposition is removed from the fermentor. The two phases are separatedby means known in the art including, but not limited to, siphoning,decantation, centrifugation, using a gravity settler, membrane-assistedphase splitting, and the like, as described above. After separation, thefermentation medium may be recycled to the fermentor or may be replacedwith fresh medium. Then, the extractant is treated to recover thebutanol product as described above. The extractant may then be recycledback into the fermentor for further extraction of the product.Alternatively, fresh extractant may be continuously added to thefermentor to replace the removed extractant. This continuous mode ofoperation offers several advantages. Because the product is continuallyremoved from the reactor, a smaller volume of organic extractantcomposition is required enabling a larger volume of the fermentationmedium to be used. This results in higher production yields. The volumeof the organic extractant composition may be about 3% to about 50% ofthe fermentor working volume; 3% to about 20% of the fermentor workingvolume; or 3% to about 10% of the fermentor working volume. It isbeneficial to use the smallest amount of extractant in the fermentor aspossible to maximize the volume of the aqueous phase, and therefore, theamount of cells in the fermentor. The process may be operated in anentirely continuous mode in which the extractant is continuouslyrecycled between the fermentor and a separation apparatus and thefermentation medium is continuously removed from the fermentor andreplenished with fresh medium. In this entirely continuous mode, thebutanol product is not allowed to reach the critical toxic concentrationand fresh nutrients are continuously provided so that the fermentationmay be carried out for long periods of time. The apparatus that may beused to carryout these modes of two-phase extractive fermentations arewell known in the art. Examples are described, for example, by Kollerupet al. in U.S. Pat. No. 4,865,973.

Batchwise fermentation mode may also be used. Batch fermentation, whichis well known in the art, is a closed system in which the composition ofthe fermentation medium is set at the beginning of the fermentation andis not subjected to artificial alterations during the process. In thismode, a volume of organic extractant composition is added to thefermentor and the extractant is not removed during the process. Theorganic extractant composition may be formed in the fermentor byseparate addition of the first and the second solvents, or the solventsmay be combined to form the extractant composition prior to the additionof the extractant composition to the fermentor. Although this mode issimpler than the continuous or the entirely continuous modes describedabove, it requires a larger volume of organic extractant composition tominimize the concentration of the inhibitory butanol product in thefermentation medium. Consequently, the volume of the fermentation mediumis less and the amount of product produced is less than that obtainedusing the continuous mode. The volume of the organic extractantcomposition in the batchwise mode may be 20% to about 60% of thefermentor working volume; or 30% to about 60% of the fermentor workingvolume. It is beneficial to use the smallest volume of extractant in thefermentor as possible, for the reason described above.

Fed-batch fermentation mode may also be used. Fed-batch fermentation isa variation of the standard batch system, in which the nutrients, forexample glucose, are added in increments during the fermentation. Theamount and the rate of addition of the nutrient may be determined byroutine experimentation. For example, the concentration of criticalnutrients in the fermentation medium may be monitored during thefermentation. Alternatively, more easily measured factors such as pH,dissolved oxygen, and the partial pressure of waste gases, such ascarbon dioxide, may be monitored. From these measured parameters, therate of nutrient addition may be determined. The amount of organicextractant composition used and its methods of addition in this mode isthe same as that used in the batchwise mode, described above.

Extraction of the product may be done downstream of the fermentor,rather than in situ. In this external mode, the extraction of thebutanol product into the organic extractant composition is carried outon the fermentation medium removed from the fermentor. The amount oforganic solvent used is about 20% to about 60% of the fermentor workingvolume; or 30% to about 60% of the fermentor working volume. Thefermentation medium may be removed from the fermentor continuously orperiodically, and the extraction of the butanol product by the organicextractant composition may be done with or without the removal of thecells from the fermentation medium. The cells may be removed from thefermentation medium by means known in the art including, but not limitedto, filtration or centrifugation. After separation of the fermentationmedium from the extractant by means described above, the fermentationmedium may be recycled into the fermentor, discarded, or treated for theremoval of any remaining butanol product. Similarly, the isolated cellsmay also be recycled into the fermentor. After treatment to recover thebutanol product, the extractant, the first solvent, and/or the secondsolvent may be recycled for use in the extraction process.Alternatively, fresh extractant may be used. In this mode the extractantis not present in the fermentor, so the toxicity of the extractant ismuch less of a problem. If the cells are separated from the fermentationmedium before contacting with the extractant, the problem of extractanttoxicity is further reduced. Furthermore, using this external mode thereis less chance of forming an emulsion and evaporation of the extractantis minimized, alleviating environmental concerns.

Methods for Production of Butanol using Extractive Fermentation with anExtractant Comprising a Dry Solvent

An improved method for the production of butanol is provided, wherein amicroorganism that has been genetically modified to produce butanol viaa biosynthetic pathway from at least one carbon source, is grown in abiphasic fermentation medium. The biphasic fermentation medium comprisesan aqueous phase and a water immiscible organic extractant compositioncomprising a dry solvent.

Isobutanol may be produced by extractive fermentation with the use of amodified Escherichia coli strain in combination with an oleyl alcohol asthe organic extractant, as disclosed in US Patent ApplicationPublication No. 2009/0305370 A1. The method yields a higher effectivetiter for isobutanol (i.e., 37 g/L) compared to using conventionalfermentation techniques (see Example 6 of US Patent ApplicationPublication No. 2009/0305370 A1). For example, Atsumi et al. (Nature451(3):86-90, 2008) report isobutanol titers up to 22 g/L usingfermentation with an Escherichia coli that was genetically modified tocontain an isobutanol biosynthetic pathway. The higher butanol titerobtained with the extractive fermentation method disclosed in U.S.Patent Application Publication No. 2009/0305370 A1 results, in part,from the removal of the toxic butanol product from the fermentationmedium, thereby keeping the level below that which is toxic to themicroorganism. It is reasonable to assume that the present extractivefermentation method employing a water-immiscible organic extractantcomposition comprising a dry solvent as defined herein would be used ina similar way and provide similar results.

Butanol produced by the method disclosed herein may have an effectivetiter of greater than 22 g per liter of the fermentation medium.Alternatively, the butanol produced by methods disclosed may have aneffective titer of at least 25 g per liter of the fermentation medium.Alternatively, the butanol produced by methods described herein may havean effective titer of at least 30 g per liter of the fermentationmedium. Alternatively, the butanol produced by methods described hereinmay have an effective titer of at least 37 g per liter of thefermentation medium. Alternatively, the butanol produced by methodsdescribed herein may have an effective titer of at least 45 g per literof the fermentation medium. Alternatively, the butanol produced bymethods described herein may have an effective titer of at least 50 gper liter of the fermentation medium. Alternatively, the butanolproduced by methods described herein may have an effective titer of atleast 60 g per liter of the fermentation medium. In some embodiments,the recovered butanol has an effective titer from about 22 g per literto about 50 g per liter, about 22 g per liter to 40 g per liter, about22 g per liter to about 30 g per liter, about 25 g per liter to about 50g per liter, about 25 g per liter to 40 g per liter, about 25 g perliter to about 30 g per liter, about 30 g per liter to about 50 g perliter, about 40 g per liter to about 50 g per liter, about 22 g perliter to about 60 g per liter, about 30 g per liter to about 60 g perliter, about 40 g per liter to about 60 g per liter, about 22 g perliter to about 80 g per liter, about 40 g per liter to about 80 g perliter, about 50 g per liter to about 80 g per liter, about 65 g perliter to about 80 g per liter. The present methods are generallydescribed below with reference to a FIGS. 1 through 7.

Referring now to FIG. 1, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol usingin situ extractive fermentation. An aqueous stream 10 of at least onefermentable carbon source is introduced into a fermentor 20, whichcontains at least one recombinant microorganism (not shown) capable ofconverting the at least one fermentable carbon source into butanol. Astream of a first dry solvent 12 and a stream of an optional secondsolvent 14 are introduced to a vessel 16, in which the solvents arecombined to form the extractant 18. A stream of the extractant 18 isintroduced into the fermentor 20, in which contacting of thefermentation medium with the extractant to form a two-phase mixturecomprising an aqueous phase and a butanol-containing organic phaseoccurs. A stream 26 comprising both the aqueous and organic phases isintroduced into a vessel 38, in which separation of the aqueous andorganic phases is performed to produce a butanol-containing organicphase 40 and an aqueous phase 42.

Referring now to FIG. 2, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol usingin situ extractive fermentation. An aqueous stream 10 of at least onefermentable carbon source is introduced into a fermentor 20, whichcontains at least one recombinant microorganism (not shown) capable ofconverting the at least one fermentable carbon source into butanol. Astream of the first dry solvent 12 and a stream of the optional secondsolvent 14 of which the extractant is comprised are introducedseparately to the fermentor 20, in which contacting of the fermentationmedium with the extractant to form a two-phase mixture comprising anaqueous phase and a butanol-containing organic phase occurs. A stream 26comprising both the aqueous and organic phases is introduced into avessel 38, in which separation of the aqueous and organic phases isperformed to produce a butanol-containing organic phase 40 and anaqueous phase 42.

Referring now to FIG. 3, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol usingin situ extractive fermentation. An aqueous stream 10 of at least onefermentable carbon source is introduced into a first fermentor 20, whichcontains at least one recombinant microorganism (not shown) capable ofconverting the at least one fermentable carbon source into butanol. Astream of the first dry solvent 12 of which the extractant is comprisedis introduced to the fermentor 20, and a stream 22 comprising a mixtureof the first dry solvent and the contents of fermentor 20 is introducedinto a second fermentor 24. A stream of the optional second solvent 14of which the extractant is comprised is introduced into the secondfermentor 24, in which contacting of the fermentation medium with theextractant to form a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase occurs. A stream 26 comprising both theaqueous and organic phases is introduced into a vessel 38, in whichseparation of the aqueous and organic phases is performed to produce abutanol-containing organic phase 40 and an aqueous phase 42.

Referring now to FIG. 4, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol inwhich extraction of the product is performed downstream of thefermentor, rather than in situ. An aqueous stream 110 of at least onefermentable carbon source is introduced into a fermentor 120, whichcontains at least one recombinant microorganism (not shown) capable ofconverting the at least one fermentable carbon source into butanol. Astream of the first dry solvent 112 and a stream of the optional secondsolvent 114 are introduced to a vessel 116, in which the solvents arecombined to form the extractant 118. At least a portion, shown as stream122, of the fermentation medium in fermentor 120 is introduced intovessel 124. A stream of the extractant 118 is also introduced intovessel 124, in which contacting of the fermentation medium with theextractant to form a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase occurs. A stream 126 comprising boththe aqueous and organic phases is introduced into a vessel 138, in whichseparation of the aqueous and organic phases is performed to produce abutanol-containing organic phase 140 and an aqueous phase 142 which maybe returned to the fermentor 120.

Referring now to FIG. 5, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol inwhich extraction of the product is performed downstream of thefermentor, rather than in situ. An aqueous stream 110 of at least onefermentable carbon source is introduced into a fermentor 120, whichcontains at least one recombinant microorganism (not shown) capable ofconverting the at least one fermentable carbon source into butanol. Astream of the first dry solvent 112 and a stream of the optional secondsolvent 114 of which the extractant is comprised are introducedseparately to a vessel 124, in which the solvents are combined to formthe extractant. At least a portion, shown as stream 122, of thefermentation medium in fermentor 120 is also introduced into vessel 124,in which contacting of the fermentation medium with the extractant toform a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase occurs. A stream 126 comprising boththe aqueous and organic phases is introduced into a vessel 138, in whichseparation of the aqueous and organic phases is performed to produce abutanol-containing organic phase 140 and an aqueous phase 142 which maybe returned to the fermentor 120.

Referring now to FIG. 6, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol inwhich extraction of the product is performed downstream of thefermentor, rather than in situ. An aqueous stream 110 of at least onefermentable carbon source is introduced into a fermentor 120, whichcontains at least one recombinant microorganism (not shown) capable ofconverting the at least one fermentable carbon source into butanol. Astream of the first dry solvent 112 of which the extractant is comprisedis introduced to a vessel 128, and at least a portion, shown as stream122, of the fermentation medium in fermentor 120 is also introduced intovessel 128. A stream 130 comprising a mixture of the first dry solventand the contents of fermentor 120 is introduced into a second vessel132. A stream of the optional second solvent 114 of which the extractantis comprised is introduced into the second vessel 132, in whichcontacting of the fermentation medium with the extractant to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase occurs. A stream 134 comprising both the aqueous andorganic phases is introduced into a vessel 138, in which separation ofthe aqueous and organic phases is performed to produce abutanol-containing organic phase 140 and an aqueous phase 142 which maybe returned to the fermentor 120.

The extractive processes described herein can be run as batch processesor can be run in a continuous mode where fresh extractant is added andused extractant is pumped out such that the amount of extractant in thefermentor remains constant during the entire fermentation process. Suchcontinuous extraction of products and byproducts from the fermentationcan increase effective rate, titer and yield.

In yet another embodiment, it is also possible to operate theliquid-liquid extraction in a flexible co-current or, alternatively,counter-current way that accounts for the difference in batch operatingprofiles when a series of batch fermentors are used. In this scenariothe fermentors are filled with fermentable mash which provides at leastone fermentable carbon source and recombinant microorganism in acontinuous fashion one after another for as long as the plant isoperating. Referring to FIG. 7, once Fermentor F100 fills with mash andmicroorganism, the mash and microorganism feed may advance to FermentorF101 and then to Fermentor F102 and then back to Fermentor F100 in acontinuous loop. The fermentation in any one fermentor begins once mashand microorganism are present together and continues until thefermentation is complete. The mash and microorganism fill time may equalthe number of fermentors divided by the total cycle time (fill, ferment,empty and clean). If the total cycle time is 60 hours and there are 3fermentors then the fill time may be 20 hours. If the total cycle timeis 60 hours and there are 4 fermentors then the fill time may be 15hours.

Adaptive co-current extraction follows the fermentation profile assumingthe fermentor operating at the higher broth phase titer can utilize theextracting solvent stream richest in butanol concentration and thefermentor operating at the lowest broth phase titer will benefit fromthe extracting solvent stream leanest in butanol concentration. Forexample, referring again to FIG. 7, consider the case where FermentorF100 is at the start of a fermentation and operating at relatively lowbutanol broth phase (B) titer, Fermentor F101 is in the middle of afermentation operating at relatively moderate butanol broth phase titerand Fermentor F102 is near the end of a fermentation operating atrelatively high butanol broth phase titer. In this case, lean extractingsolvent (S), with minimal or no extracted butanol, can be fed toFermentor F100, the “solvent out” stream (S′) from Fermentor F100 havingan extracted butanol component can then be fed to Fermentor F101 as its“solvent in” stream and the solvent out stream from F101 can then be fedto Fermentor F102 as its solvent in stream. The solvent out stream fromF102 can then be sent to be processed to recover the butanol present inthe stream. The processed solvent stream from which most of the butanolis removed can be returned to the system as lean extracting solvent andwould be the solvent in feed to Fermentor F100 above.

As the fermentations proceed in an orderly fashion the valves in theextracting solvent manifold can be repositioned to feed the leanestextracting solvent to the fermentor operating at the lowest butanolbroth phase titer. For example, assume (a) Fermentor F102 completes itsfermentation and has been reloaded and fermentation begins anew, (b)Fermentor F100 is in the middle of its fermentation operating atmoderate butanol broth phase titer and (c) Fermentor F101 is near theend of its fermentation operating at relatively higher butanol brothphase titer. In this scenario the leanest extracting solvent would feedF102, the extracting solvent leaving F102 would feed Fermentor F100 andthe extracting solvent leaving Fermentor F100 would feed Fermentor F101.

An advantage of operating this way can be to maintain the broth phasebutanol titer as low as possible for as long as possible to realizeimprovements in productivity. Additionally, it can be possible to dropthe temperature in the other fermentors that have progressed furtherinto fermentation that are operating at higher butanol broth phasetiters. The drop in temperature can allow for improved tolerance to thehigher butanol broth phase titers.

Having described a variety of techniques, approaches, systems and soforth that can implement dry solvents, including multiple solvents,multiple solvent extraction techniques are now be described inadditional detail. It should be apparent that the techniques,approaches, compositions, and so on described in conjunction with asolvent mixture, can implement the principles previously described andvice versa. Additionally, while multiple solvent systems including drysolvents are described, it is to be apparent that comparatively “wet”solvent systems can benefit from the principles of this disclosure.

Extraaction of Butanol using a Solvent Mixture

In embodiments, a solvent mixture is used to extract alcohol from anaqueous solution. For example, a solvent mixture and fermentation brothare contacted together to extract butanol, such as isobutanol from thebroth. The contacting can be performed internally (internal to afermentor) externally (e.g., via a cooling loop), or a combinationthereof, and so forth as described above. As also is described above, afermentation broth can include, but is not limited to, fermentationproducts, fermentation solids, unfermented carbon substrate (e.g.,sugar), microorganisms (alive, dead, intended, unintended), nutrients(e.g., mineral nutrients use by a microorganism to produce alcohol), andso forth.

In examples, the solvent mixture includes a first solvent and a secondsolvent. Optionally, additional solvents (three or more), additives, andso forth to promote efficient extraction can be included in the solventmixture as contemplated by one of ordinary skill in the art.

The individual solvents can be selected so the resulting mixtureexhibits properties that increase extraction efficiency, improveextraction selectivity (e.g., preferentially extracting, for example, atarget alcohol (isobutanol) in comparison to other compound such aswater), or is an anti-solvent for nutrients. Additional examplesinclude, but are not limited to, minimizes moisture content (tendency ofthe solvent to dissolve water, or wetness), exhibits goodhydrophobicity, the ability to be separated from the target alcohol bydistillation, it is a good solvent for inhibitory impurities orco-products, the solvents are economically viable, environmentalconsiderations, and the like. In embodiments, hydrophobicity isexpressed as log P, which is the log of the partition coefficient of thesolvent or solvent mixture in a mix of solvent/octanol/water. Thus, logP can be expressed as the base ten logarithm of the ratio of the totalmolar concentration of the solvent(s) in the organic phase divided bythe total molar concentration of the solvent(s) in the aqueous phase inthe presence of octanol, e.g., the sum of all solvents.

Other relevant solvent mixture properties that can be tailored include,but are not limited to, low toxicity/biocompatibility to a microorganismthat is capable of producing the alcohol, low tendency to extractnutrients (e.g., nutrients used by a microorganism to produce alcohol),boiling point, compatibility with other solvents to be included in thesolvent mixture, thermal stability, low volatility, and so on. Examplenutrients include minerals, and vitamins. A solvent's affinity to aminoacids, proteins, peptides, and peptones also can be considered. In someembodiments, one or more of the solvents or the solvent mixture is usedto transport nutrients to a fermentation broth. In examples such asthis, the solvent mixture can contact nutrients prior to contacting thebroth. Accordingly, the nutrients can be exchanged with the broth so thenutrients enter the broth and the alcohol enters the solvent mixture.Other relevant properties include a solvent's affinity to impuritiesthat inhibit the microorganism. For example, a solvent mixture includesa solvent that has a high affinity to a compound that is produced duringfermentation, but is toxic to a microorganism generating the productalcohol. The ability to separate the solvents one from another may beconsidered, if for example, a solvent is provided as an offtake afteruse. COFA can be purified to separate any co-solvents and provided as anoff-take product.

A solvent can be selected because it is effective for removing acontaminate (e.g., butyric acid) that is toxic to the microorganism.While maximizing beneficial properties is preferred, trade-offs can bemade to avoid or minimize non-preferred properties. For example, whilesome solvents have high butanol affinity (Kd) they can exhibit highmoisture content (wetness), and/or are toxic to a microorganism. Othersolvents are considered to be dry (low moisture), have goodbiocompatibility (high log P), but exhibit low or poor Kd.

In some implementations, the solvent mixture exhibits one or moreproperties that are not indicated by a linear molar combination of thefirst and second solvents. Some solvent mixtures, for example, exhibitproperties that are not indicated based on the properties of theindividual solvents and the mole factions of the individual solvents.For example, it may be expected that a fifty/fifty (50/50) ratio of afirst and second solvent for a particular characteristic (e.g.,hydrophobicity) would behave as if the solvent mixture's correspondingproperty or characteristic was fifty percent (50%) that of the firstsolvent and fifty percent (50%) that of the second solvent. In someinstances, the solvent mixture exhibits a property that is influenced toa comparatively greater degree by one of the solvents in the mixturethan the other solvent. The foregoing is also applicable to solventmixtures including more than two solvents. In some instances, thisdeparture from that indicated by a linear combination is due tointermolecular interactions between the solvents in the solvent mixture.Examples include, but are not limited to, polarity, existence ofhydrogen bonding, van der Waals forces, e.g., London forces, and thelike. This departure from predicted behavior can be graphicallyrepresented (generally and in a simplified fashion) by the diagram shownin FIG. 15.

As can be seen in FIG. 15, with respect to solvent properties, a binarysolvent mixture can depart from that which is indicated by a linearcombination of the first and second solvent. As illustrated, theproperty may depart from that which is expected (e.g., the “Linearcombination”) from the individual solvents based on their respectivemole fractions as generally illustrated above. The property orcharacteristic can be beneficial (e.g., good selectivity in a butanolextraction, high affinity for butanol) or it may exhibit a negativeproperty (e.g., exhibit poor hydrophobicity). Thus, a solvent mixturecan depart from that indicated by the linear combination (generally)along the two curved lines. For example, mixing a first solvent that isdry (low moisture) and biocompatible (high log P) with a microorganism(butanologen) with a second wet solvent that has a high Kd can result ina solvent mixture that is efficient at extracting butanol fromfermentation broth and exhibits good biocompatibility with butanalogensin the broth. In this way, a solvent mixture can be tailored to exhibita synergistic effect, a favorable effect that is not indicated by alinear combination of the first and second solvents. For example, asolvent mixture of isohexadecane and isododecanol can exhibit goodhydrophobicity and may be generally non-toxic (e.g., biocompatible) witha butanologen in comparison to a solvent mixture of thymol and COFA.

The properties of a solvent mixture are sometimes described by a complexfunction of the component mole fractions. For example, the naturallogarithm of the activity coefficient of a component in a non-idealliquid mixture can be expressed by an empirical model of solutionbehavior such as that provided by the Margules equation. In thisinstance, the natural logarithm of the activity coefficient of any oneof the components is a third order polynomial function of the molefractions of all of the components in the solvent mixture.

When distributing isobutanol, for example, between two contactingimmiscible non-ideal liquid mixtures (e.g., an aqueous phase and anorganic phase), an equilibrium molar partition coefficient can beequated to a ratio of the activity coefficient of isobutanol in onemixture (e.g., aqueous phase) to the activity coefficient of isobutanolin the other mixture (e.g., organic phase). The natural logarithm of themolar partition coefficient can therefore be equated to the differencebetween the natural logarithm of the two activity coefficients whichwill follow a third order polynomial function. One skilled in the artcan expect that properties such as Kd and log P of a mixture can followsimilarly if not more complex functions of the component mole fractions.Describing the equilibrium moisture content of a non-ideal liquidmixture as a function of the component mole fractions can be even morecomplex.

While the solvent mixture or portions thereof can be immiscible inwater, e.g., on the order of 10⁻⁷, in some instances one or more of thesolvents can be comparatively weakly miscible in water. It is also to beappreciated, that water can be miscible (e.g., somewhat miscible) in thesolvent mixture and/or miscible in one or more solvents included in thesolvent mixture. COFA in some examples can absorb moisture, or water,such that the COFA is “wet”. A solvent's tendency to absorb moisture,e.g., act as a solvent, in some examples, can differ from that solvent'sability to solvate in water, e.g., its hydrophobicity. A table ofvarious solvents and their respective characteristics is reproduceddirectly below.

TABLE 1 Example solvent properties Moisture Solvent LogP Kd Content (wt%) Corn oil 19 0.25 0.70 FABE 9.4 1.4 0.18 COFA 6.5 3 0.702-ethylhexanol 2.8 7.8 2 Carvacrol 3.3 15.6 2 Tetrabutylurea 6.6 7.4 0.9Isododecanol 4.4 5.4 0.2 Tributylphosphate 4.3 9.8 6.7 Isododecane 6.20.25 <0.01 Isohexadecane 8.0 0.2 <0.01 Triisopropylbenzene 6.2 0.65<0.01Confirmation of Isobutanol Production

The presence and/or concentration of isobutanol in the culture mediumcan be determined by a number of methods known in the art (see, forexample, U.S. Pat. No. 7,851,188, incorporated by reference). Forexample, a specific high performance liquid chromatography (HPLC) methodutilizes a Shodex SH-1011 column with a Shodex SHG guard column, bothmay be purchased from Waters Corporation (Milford, Mass.), withrefractive index (RI) detection. Chromatographic separation is achievedusing 0.01 M H₂SO₄ as the mobile phase with a flow rate of 0.5 mL/minand a column temperature of 50° C. Isobutanol has a retention time of46.6 min under the conditions used.

Alternatively, gas chromatography (GC) methods are available. Forexample, a specific GC method utilizes an HP-INNOWax column (30 m×0.53mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.),with a flame ionization detector (FID). The carrier gas is helium at aflow rate of 4.5 mL/min, measured at 150° C. with constant headpressure; injector split is 1:25 at 200° C.; oven temperature is 45° C.for 1 min, 45 to 220° C. at 10° C./min, and 220° C. for 5 min; and FIDdetection is employed at 240° C. with 26 mL/min helium makeup gas. Theretention time of isobutanol is 4.5 min.

While various embodiments of the present invention have been describedherein, it should be understood that they have been presented by way ofexample only, and not limitation. It will be apparent to persons skilledin the relevant art that various changes in form and detail can be madetherein without departing from the spirit and scope of the invention.Thus, the breadth and scope of the present invention should not belimited by any of the above-described exemplary embodiments, but shouldbe defined only in accordance with the claims and their equivalents.

All publications, patents, and patent applications mentioned in thisspecification are indicative of the level of those skilled in the art towhich this invention pertains, and are herein incorporated by referenceto the same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating embodimentsof the invention, are given by way of illustration only. From the abovediscussion and these Examples, one skilled in the art can ascertain theessential characteristics of this invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the invention to adapt it to various uses and conditions.

Some of the examples described herein are demonstrated usingcomputational modeling such as Aspen modeling (see, e.g., U.S. Pat. No.7,666,282). For example, the commercial modeling software Aspen Plus®(Aspen Technology, Inc., Burlington, Mass.) may be used in conjunctionwith physical property databases such as DIPPR and UNIFAC, availablefrom American Institute of Chemical Engineers, Inc. (New York, N.Y.) todevelop an Aspen model for an integrated butanol fermentation,extraction, purification, and water management process. This processmodeling can perform many fundamental engineering calculations, forexample, mass and energy balances, vapor/liquid equilibrium, andreaction rate computations. In order to generate an Aspen model,information input may include, for example, experimental data, watercontent and composition of feedstock, temperature for mash cooking andflashing, saccharification conditions (e.g., enzyme feed, starchconversion, temperature, pressure), fermentation conditions (e.g.,microorganism feed, glucose conversion, temperature, pressure),degassing conditions, solvent columns, pre-flash columns, condensers,evaporators, centrifuges, and so forth.

Example 1A: Biocompatibility of Triisobutylene with Ethanologen

Into each of two 125 ml shake flasks, 20 ml of an aqueous culture mediumcontaining glucose at a concentration of 21 g/liter and a 0.5 ODsuspension of a naturally occurring ethanologen yeast strain was added.A volume of 10 ml of triisobutylene (Tokyo Chemical Industry Co., >90%mixture of isomers, with an estimated log P of 5.8) was added on top ofthe aqueous suspension of one of the flasks. These flasks were placed inan incubating oven controlled at 32° C. and continuously shaken. After 4hours, the aqueous phases of both flasks were analyzed and found tocontain less than 0.1 g/liter glucose. No significant difference inglucose uptake was observed between the two flasks.

Example 1B: Biocompatibility of Triisobutylene with Isobutanolagen

Two 125 ml flasks were individually prepared with twenty milliliters (20ml) of an aqueous culture medium containing glucose at a concentrationof twenty-eight grams per liter (28 g/L) to which a zero point five(0.5) OD suspension of a genetically modified isobutanolagen yeaststrain (PNY2141) was added. Ten milliliters (10 ml) of triisobutylene(Tokyo Chemical Industry Co., >90% mixture of isomers, with an estimatedlog P of 5.8) was added on top of the aqueous suspension to one of theflasks. The flasks were placed in an incubating oven controlled atthirty-two degrees Celsius (32° C.) and continuously shaken. The glucoseconcentration and OD were monitored for each of the two flasks. Table 2,reproduced below indicates the results from the monitored flasks.

Growth was monitored by measuring OD and glucose concentration. Someimpedance to growth was observed in the flask containing thetriisobutylene.

TABLE 2 OD and glucose concentrations for isobutanol extractionwithtriisobutylene No Solvent With solvent Glucose, gpl Glucose, gpl Time,hr OD Grams per Liter OD Grams per Liter 0 0.5 28 0.5 28 6 0.7 26 0.6 2710 1.0 24 0.8 26 19 3.7 12 1.6 22 23 4.5 2 2.1 21 30 4.4 0 2.8 14 35 4.30 3.4 10

Example 2: Biocompatibility of a Triisobutylene-COFA Mixture

Into each of two 125 ml flasks, 20 ml of an aqueous culture mediumcontaining glucose at a concentration of 32 g/liter and a 0.5 ODsuspension of a genetically modified isobutanologen yeast strain wasadded. A volume of 10 ml of a 50% mixture of triisobutylene (TokyoChemical Industry Co., >90% mixture of isomers, with an estimated log Pof 5.8) and corn oil fatty acid was added on top of the aqueoussuspension of one of the flasks. These flasks were placed in anincubating oven controlled at 32° C. and continuously shaken. Growth wasmonitored by measuring OD. No significant difference in growth wasobserved between the two flasks as shown in Table 3.

TABLE 3 OD values for an isobutanologen grown in the presence andabsence of a 50:50 mixture of triisobutylene and corn oil fatty acid(COFA). Time (hr) OD with No Solvent OD with solvent 0 0.5 0.5 7 0.9 0.913 1.5 1.6 23 3.7 3.1 29 4.1 4.0 37 4.3 4.3 50 5.0 5.0 60 5.4 5.5

Example 3: Biocompatibility of Isododecane

Into each of two 125 ml flasks, 20 ml of an aqueous culture mediumcontaining glucose at a concentration of 32 g/liter and a 0.5 ODsuspension of a genetically modified isobutanolagen yeast strain wasadded. A volume of 10 ml of isododecane (Alfa Aesar Technical grademixture of isomers, with an estimated log P of six point two (6.2)) wasadded on top of the aqueous suspension of one of the flasks. Theseflasks were placed in an incubating oven controlled at 32° C. andcontinuously shaken. Growth was monitored by measuring glucose uptakeand OD. No significant difference in growth was observed between the twoflasks as shown in Table 3, directly below.

TABLE 3 OD and glucose values for an isobutanologen grown in thepresence/absence of isododecane No Solvent With Solvent Time (hr) ODGlucose (g/L) OD Glucose (g/L) 0 0.5 32 0.5 32 7 0.9 32 0.9 32 13 1.5 251.6 25 23 4.8 5 4.9 5 29 4.1 0.1 5.0 0.1

Example 4: Isobutanol Partitioning Between Triisobutylene and Water

Into a small round bottom flask, 10 ml of an aqueous solution containingisobutanol at a concentration of 6 wt % and 1 ml of triisobutylene(Tokyo Chemical Industry Co., >90% mixture of isomers, with an estimatedlog P of 5.8) were combined. The liquids were mixed thoroughly and thencentrifuged to separate into organic and aqueous layers. A sample of theorganic layer was analyzed by gas chromatography and found to contain9.03 wt % (or 18.39 mole %) isobutanol. A sample of the aqueous layerwas analyzed by gas chromatography and found to contain 5.28 wt % (or1.33 mole %) isobutanol. The mass partitioning coefficient is calculatedto be 1.71 and the molar partitioning coefficient was calculated to be13.8.

Example 5A: Isobutanol Partitioning Between Isododecane and Water (FirstCondition Set)

Into a small round bottom flask, 10 ml of an aqueous solution containingisobutanol (Sigma-Aldrich Co. LLC, St. Louis, Mo., USA, reagent grade)at a concentration of 6 wt % and 1 ml of isododecane (Alfa AesarTechnical grade mixture of isomers, with an estimated log P of six pointtwo (6.2)) were combined. The liquids were mixed thoroughly and thencentrifuged to separate into organic and aqueous layers. A sample of theorganic layer was analyzed by gas chromatography and found to contain8.31 wt % (or 17.24 mole %) isobutanol. A sample of the aqueous layerwas analyzed by gas chromatography and found to contain 5.45 wt % (or1.39 mole %) isobutanol. The mass partitioning coefficient is calculatedto be 1.525 and the molar partitioning coefficient was calculated to be12.4.

Example 5B: Isobutanol Partitioning Between Isododecane and Water(Second Condition Set)

Into a sample vial, three grams (3 g) of an aqueous solution containingisobutanol at a concentration of two point zero weight percent (2.0 wt%) and three (3 g) of isododecane were combined. The liquids were mixedthoroughly and then centrifuged to separate into organic and aqueouslayers. A sample of the aqueous layer was analyzed by HPLC and found tocontain one point six zero weight percent (1.60 wt %) isobutanol and theorganic layer was calculated by mass balance to contain zero point fourzero weight percent (0.40 wt %) isobutanol. The mass partitioningcoefficient was calculated to be zero point two five (0.25).

Example 6A: Biocompatibility of 1,3-Diisopropylbenzene

Into a 125 ml flask, twenty milliliters (20 ml) of an aqueous culturemedium containing glucose at a concentration of thirty grams per liter(30 g/liter) and a one point zero 1.0 OD suspension of a geneticallymodified isobutanolagen yeast strain (PNY2141) was added. A volume of 10ml of 1,3-diisopropylbenzene (Sigma-Aldrich Co. LLC, St. Louis, Mo.,USA, (Aldrich) reagent grade, with an estimated log P of four point nine(4.9)) was added on top of the aqueous suspension. The flask was placedin an incubating oven controlled at thirty-two degrees Celsius (32° C.)and continuously shaken. Growth was monitored by measuring glucose. Nosignificant consumption of glucose was observed. Results of thisanalysis are reported in Table 4, directly below.

TABLE 4 Glucose Levels for 1,3-diisopropylbenzene Time (hours) Glucose(grams per liter, gpl) 0 30 6 30 11 30 16 28 24 29

Example 6B: Biocompatibility of 1,3,5-Triisopropylbenzene

In this example, a 1.0 OD suspension of a genetically modifiedisobutanolagen yeast strain PNY2310 is added to a 125 ml flask including20 ml of an aqueous culture medium containing glucose at a concentrationof 29 g/liter. Ten milliliters (10 ml) of 1,3,5-triisopropylbenzene(Sigma-Aldrich, reagent grade, with an estimated log P of 6.2) was addedon top of the aqueous suspension. The flask including the sample wasmaintained at thirty-two degrees Celsius (32° C.) in an incubating ovenwith continuous shaking Glucose was measured to monitor its consumption.No significant inhibition in glucose consumption was observed with theinclusion of the triisopropylbenzene. Table 5, directly below, indicatesthe results. As can be observed, addition of an isopropyl group made asignificant impact. This impact may be attributed to the presence of thepropyl group.

TABLE 5 Glucose Levels for 1,3,5-Triisopropylbenzene Time (hours)Glucose (grams per liter, gpl) 0 29 6 25 11 20 16 12 24 1

Example 7: Isobutanol Partitioning Between 1,3,5-Triisopropylbenzene andWater

In an example, five point two grams (5.2 g) of triisopropylbenzene(Sigma-Aldrich, reagent grade) and an aqueous solution containingisobutanol at a concentration of two point four weight percent (2.4 wt%) were combined in a sample vial. The 1,3,5-triisopropylbenzene andaqueous solution were thoroughly mixed and centrifuged to separate theorganic and aqueous layers from one another. Analysis of a sampleobtained from the organic layer, e.g., the layer containing1,3,5-triisopropylbenzene, was analyzed using gas chromatography asdescribed in the section captioned “confirmation of isobutanolproduction.” This analysis indicated the organic lay included one pointone eight weight percent (1.18 wt %) isobutanol. A sample obtained fromthe aqueous layer was analyzed using high pressure liquid chromatography(HPLC) as described in the section captioned “confirmation of isobutanolproduction.” This analysis indicated that the aqueous layer containedone point eight four weight percent (1.84 wt %) isobutanol. The masspartitioning coefficient was calculated from the weight percentages inthe organic and aqueous layers, the mass partitioning coefficient waszero point six four (0.64).

Example 8: Isobutanol Partitioning Between1,3,5-Triisopropylbenzene/Thymol Blend and Water

In an example, zero point two five grams (0.25 g) of thymol(Sigma-Aldrich Co. LLC, St. Louis, Mo., USA, reagent grade) was combinedwith ten point two five grams (10.25 g) of aqueous solution thatcontained isobutanol at a concentration of two point four weightpercentage (2.4 wt %) in a sample vial and four point seven five grams(4.75 g) of 1,3,5-triisopropylbenzene. The thymol,1,3,5-triisopropylbenzene, and aqueous solutions were thoroughly mixedand centrifuged to form an organic layer and an aqueous layer. Gaschromatograph was used to analyze a sample obtained from the organiclayer. The organic layer sample was determined to contain two pointthree zero weight percent (2.30 wt %) of isobutanol as described in thesection captioned “confirmation of isobutanol production.” A sample fromthe aqueous layer was analyzed using HPLC. The aqueous sample wasdetermined to contain one point three seven weight percent (1.37 wt %)isobutanol as described in the section captioned “confirmation ofisobutanol production.” The mass partitioning coefficient was calculatedfrom the weight percentages in the organic and aqueous layers, the masspartitioning coefficient was one point seven zero (1.70).

In embodiments, thymol exhibits fungicidal properties. Thymol can beused to break down yeast cells resulting in lysis of the cells, forexample. In some embodiments, thymol exhibited in vitro antifungalproperties to Sacchromyces Cerevisiae at 1.5 mM (MIC, minimum inhibitoryconcentration) concentration while the MFC (minimum fungalconcentration) was 1.8 mM. Examples of thymol's fungicidal propertiesare reported in Bennis et al., Surface Alteration of SacchromycesCerevisiae Induced by Thymol and Eugenol, 38 Letters in AppliedMicrobiology 454-458 (2004), which is hereby incorporated by referencein its entirety.

Thymol may be of interest as its partition coefficient (Kd) forwater/butanol/solvent (e.g., is calculated from an equilibrium ternarymixture of water/butanol/solvent) is greater than twenty-five (>25) andis, comparatively, much less hindered than BHT. Thymol is naturallyoccurring (main extract of thyme) and considered GRAS (generallyrecognized as safe). It is used as a nontoxic insect repellant. It canserve as an antioxidant. It also is antibacterial but was shown to beequally damaging to yeast. In embodiments a recombinate microorganism(such as genetically modified butanologen) can be evolved in thepresence of thymol and/or a COFA thymol mixture so the microorganism canoutgrow yeast or bacteria that naturally occur in the same environment.In embodiments such as this, the COFA thymol mixture can exhibit high Kdand be implemented as an extraction solvent that is oxidatively stable.The log P for thymol is 3.3. Accordingly, the biocompatibility of thymolis sufficient so in some embodiments thymol is included in the mixturewith COFA at or approximately at or below ten percent (10%) by volume.In some examples, a ten percent thymol/COFA mix has a Kd of four pointeight (4.8). For comparison, some thymol/COFA mixes under ten percent(10%) by volume exhibit a Kd of at or approximately at three (3).Accordingly, thymol/COFA exhibits greater synergy with respect to Kd incomparison to thymol/corn oil.

Example 9: Biocompatibility of Tetrabutylurea

Into each of two 125 ml flasks, twenty milliliter (20 ml) of an aqueousculture medium containing glucose at a concentration of twenty-eightgrams per liter (28 g/L) and a 0.5 OD suspension of a geneticallymodified isobutanolagen yeast strain PNY2310 was added. A volume of tenmilliliters (10 ml) of tetrabutylurea ((Tokyo Chemical Industry Co.,estimated log P of six point six (6.6)) was added on top of the aqueoussuspension of one of the flasks. These flasks were placed in anincubating oven controlled at thirty-two degrees Celsius (32° C.) andcontinuously shaken. Growth was monitored by measuring glucoseconcentration. No significant difference was observed between the twoflasks. The results are shown in Table 6, directly below.

TABLE 6 Glucose concentration for an isobutanologen grown in thepresence/absence of tetrabutylurea No Solvent With Solvent Time (hr)Glucose (g/L) Glucose (g/L) 0 28 28 4 25 25 8 14 14 12 1 0

Example 10A: Isobutanol Partitioning Between Tetrabutylurea and Water

Into a sample vial, five grams (5 g) of an aqueous solution containingisobutanol (Sigma-Aldrich Co. LLC, St. Louis, Mo., USA, reagent grade)at a concentration of two point zero weight percent (2.0 wt %) and twopoint five (2.5 g) of tetrabutylurea ((Tokyo Chemical Industry Co.,estimated log P of six point six (6.6)) were combined. The liquids weremixed thoroughly and then centrifuged to separate into organic andaqueous layers. A sample of the aqueous layer was analyzed by HPLC andfound to contain zero point four one seven weight percent (0.417 wt %)isobutanol and the organic layer was calculated by mass balance tocontain three point zero six weight percent (3.06 wt %) isobutanol. Themass partitioning coefficient was calculated to be seven point threefive (7.35).

Example 10B: Isobutanol Partitioning Between anIsododecane/Tetrabutylurea Blend and Water

Into a sample vial, three grams (3 g) of an aqueous solution containingisobutanol (Sigma-Aldrich Co. LLC, St. Louis, Mo., USA, reagent grade)at a concentration of two point zero weight percent (2.0 wt %), onepoint five grams (1.5 g) of isododecane (Alfa Aesar Technical grademixture of isomers, with an estimated log P of six point two (6.2)) andone point five grams (1.5 g) of tetrabutylurea (Tokyo Chemical IndustryCo., estimated log P of six point six (6.6)) were combined. The liquidswere mixed thoroughly and then centrifuged to separate into organic andaqueous layers. A sample of the aqueous layer was analyzed by HPLC andfound to contain zero point four six weight percent (0.46 wt %)isobutanol and the organic layer was calculated by mass balance tocontain one point five three weight percent (1.53 wt %) isobutanol. Themass partitioning coefficient was calculated to be three point threethree (3.33). See also Example 5B for isobutanol partitioning betweenisododecane and water.

Example 11A: Isobutanol Partitioning Between 2,6,8-Trimethyl-4-Nonanol(an Isododecanol) and Water

Into a sample vial, five grams (5 g) of an aqueous solution containingisobutanol (Sigma-Aldrich Co. LLC, St. Louis, Mo., USA, reagent grade)at a concentration of two point zero weight percent (2.0 wt %) and twopoint five grams (2.5 g) of 2,6,8-trimethyl-4-nonanol (Pfaltz & Bauer,Inc., Waterbury, Conn.) were combined. The liquids were mixed thoroughlyand then centrifuged to separate into organic and aqueous layers. Asample of the aqueous layer was analyzed by HPLC and found to containzero point five three weight percent (0.53 wt %) isobutanol and theorganic layer was calculated by mass balance to contain two point eightweight percent (2.87 wt %) isobutanol. The mass partitioning coefficientwas calculated to be five point four two (5.42).

Example 11B: Isobutanol Partitioning Between anIsododecane/2,6,8-Trimethyl-4-Nonanol Blend and Water

Into a sample vial, five grams (5 g) of an aqueous solution containingisobutanol (Sigma-Aldrich Co. LLC, St. Louis, Mo., USA, reagent grade)at a concentration of two point zero weight percent (2.0 wt %), fourpoint five grams (4.5 g) of isododecane (Alfa Aesar Technical grademixture of isomers, with an estimated log P of six point two (6.2)) andzero point five grams (0.5 g) of 2,6,8-trimethyl-4-nonanol (Pfaltz &Bauer, Inc., Waterbury, Conn.) were combined. The liquids were mixedthoroughly and then centrifuged to separate into organic and aqueouslayers. A sample of the aqueous layer was analyzed by HPLC and found tocontain one point three three weight percent (1.33 wt %) isobutanol andthe organic layer was calculated by mass balance to contain two pointtwo eight weight percent (2.28 wt %) isobutanol. The mass partitioningcoefficient was calculated to be one point seven one (1.71). See alsoExample 5B for isobutanol partitioning between isododecane and water.

Example 12: Biocompatibility of Tris(2-Ethylhexyl) Phosphate

Into a 125 ml flask, twenty milliliters (20 ml) of an aqueous culturemedium containing glucose at a concentration of seventeen grams perliter (17 g/L) and a one point one (1.1) OD suspension of an ethanol redyeast strain was added. A volume of ten milliliters (10 ml) oftris(2-ethylhexyl) phosphate (Sigma-Aldrich Co. LLC, St. Louis, Mo.,USA, reagent grade, with an estimated log P of ten point one (10.1)) wasadded on top of the aqueous suspension. The flask was placed in anincubating oven controlled at thirty-two degrees Celsius (32° C.) andcontinuously shaken. Growth was monitored by measuring glucose. Nosignificant inhibition in glucose consumption was observed. The resultsare reported in Table 7, reproduced directly below.

TABLE 7 Glucose Levels for tris(2-Ethylhexyl) phosphate Time (hours)Glucose (grams per liter, gpl) 0 17 2.5 11.5 5 0

Example 13: Isobutanol Partitioning Between Triisobutylene and Water

Into a sample vial, five grams (5 g) of an aqueous solution containingisobutanol at a concentration of two point zero weight percent (2.0 wt%) and five grams (5 g) of triisobutylene (Tokyo Chemical Industry Co.)were combined. The liquids were mixed thoroughly and then centrifuged toseparate into organic and aqueous layers. A sample of the aqueous layerwas analyzed by HPLC and found to contain one point five three weightpercent (1.53 wt %) isobutanol and the organic layer was calculated bymass balance to contain zero point four seven weight percent (0.47 wt %)isobutanol. The mass partitioning coefficient was calculated to be zeropoint three one (0.31). See also Example 4 for this system underdifferent conditions.

Example 14: Isobutanol Partitioning Between Tributyl Phosphate and Water

Into a sample vial, five grams (5 g) of an aqueous solution containingisobutanol at a concentration of two point zero weight percent (2.0 wt%) and two point five grams (2.5 g) of tributyl phosphate (Sigma-AldrichCo. LLC, St. Louis, Mo., USA, reagent grade) were combined. The liquidswere mixed thoroughly and then centrifuged to separate into organic andaqueous layers. A sample of the aqueous layer was analyzed by HPLC andfound to contain zero point three three weight percent (0.33 wt %)isobutanol and the organic layer was calculated by mass balance tocontain three point two two weight percent (3.22 wt %) isobutanol. Themass partitioning coefficient was calculated to be nine point eight(9.8).

Example 15: Isobutanol Partitioning Between DEET and Water

Into a sample vial, one gram (1 g) of an aqueous solution containingisobutanol at a concentration of two point zero weight percent (2.0 wt%) and zero point five grams (0.5 g) of DEET (Sigma-Aldrich Co. LLC, St.Louis, Mo., USA, reagent grade) were combined. The liquids were mixedthoroughly and then centrifuged to separate into organic and aqueouslayers. A sample of the aqueous layer was analyzed by HPLC and found tocontain zero point three four weight percent (0.34 wt %) isobutanol andthe organic layer was calculated by mass balance to contain three pointone seven weight percent (3.17 wt %) isobutanol. The mass partitioningcoefficient is calculated to be nine point two (9.2).

Example 16: Isobutanol Partitioning Between Carvacrol and Water

Into a sample vial, five grams (5 g) of an aqueous solution containingisobutanol at a concentration of two point zero weight percent (2.0 wt%) and two point five grams (2.5 g) of carvacrol (Sigma-Aldrich Co. LLC,St. Louis, Mo., USA, reagent grade) were combined. The liquids weremixed thoroughly and then centrifuged to separate into organic andaqueous layers. A sample of the aqueous layer was analyzed by HPLC andfound to contain zero point two two weight percent (0.22 wt %)isobutanol and the organic layer was calculated by mass balance tocontain three point four two weight percent (3.42 wt %) isobutanol. Themass partitioning coefficient is calculated to be fifteen point five(15.5).

Example 17: Solubility of Water in Solvents

By way of example, a study was conducted using Aspen Plus simulationsoftware (Aspen Technology, Inc., Burlington, Mass., U.S.A.) to comparethree solvents. The solvents studied were corn oil fatty acid (COFA),oleyl alcohol and isolauryl alcohol (ISOLAUR). Table 3 shows theequilibrium water content in the solvents in the presence of about twopercent by weight (2 wt %) butanol at about thirty-two degrees Celsius(32° C.).

TABLE 8 Water content of different solvents Solvent Water Content, wt %COFA 0.84 Oleyl 1.5 ISOLAUR 2.0

In this example a mixture of organic solvent, butanol and water waspreheated under pressure before distilling to remove butanol from thesolvent. In all cases, the butanol concentration in the solvent wasabout two percent by weight (2 wt %) and the feed temperature to theheat exchanger was about thirty-two degrees Celsius (32° C.). Thetemperature of the preheater was varied between ninety degrees Celsius(90° C.) and one hundred thirty degrees Celsius (130° C.) prior toentering the distillation unit. The variable of interest was therequired heat duty to raise the mixture to the preheat temperature.Results as shown in FIG. 9, demonstrated that, from the selectedsolvents, a higher water content in the feed led to a greater heatrequirement to reach a given temperature. For each composition the watercontent was fixed at its equilibrium value presented in the Table 8. Ineach mixture, the mass of butanol and solvent was identical.

For a given solvent in a mixture of solvent, water and butanol, therequired heat duty to reach a given temperature is sensitive to theamount of water present. A separate study was conducted using Aspen Plussimulation software to show the required heat duty to reach a giventemperature as a function of water content in a mixture oftriisobutylene, butanol, and water. Water content was varied from zeropercent (0%) to about three percent (3%) with a fixed heat exchangertemperature of one hundred degrees Celsius (100° C.). The additionalwater load in the stream led to an increase in required heat exchangerduty as demonstrated in FIG. 10.

Discussion of Examples

In embodiments, the base solvent is chosen to be dry, but it may notexhibit sufficient butanol affinity, e.g., isobutanol affinity. The basesolvent in these embodiments can be chosen to have excessbiocompatibility. A solvent's biocompatibility can be correlated to thesolvent's log P value. In some embodiments, it is preferable to form amix of solvents where one solvent (e.g., a base solvent or firstsolvent) exhibits high biocompatibility while the other solvent (e.g., asecond solvent) exhibits other characteristics (e.g., high affinity tobutanol, exhibit a synergistic effect in a solvent mix), but it mayexhibit lower or poor biocompatibility. Solvents can be selected basedon the mixture's properties that can differ from those of the solventsor those predicted from the properties of the solvents forming themixture according to each solvent's molar ratio in the mixture. For somebutanol-producing organisms, a maximum log P of six (6) or ofapproximately six (6) indicates biocompatible, such as biocompatibilityfor a butanologen (e.g., a microorganism genetically modified to producebutanol). This is to say that for some fermentation systemshydrophobicity is associated with biocompatibility or toxicity.

A log P of six (6) may correspond to an equilibrium, saturatedconcentration of solvent in the aqueous phase of zero point two partsper million (0.2 ppm) or approximately zero point two parts per million(0.2 ppm). At this concentration, the solvent may be sufficientlydispersed in the aqueous phase to avoid interfering with themicroorganism's metabolism of sugar (glucose) to alcohol. Increasing theconcentration of solvent in the aqueous phase, to for example zero pointthree parts per million (0.3 ppm) or approximately zero point threeparts per million (0.3 ppm) can have a detrimental impact on amicroorganism's ability to produce alcohol. Additionally, for example, asolvent can hinder fermentation if it has a high affinity for nutrientsused by the microorganism in fermentation. A solvent may hinder amicroorganism by interfering with the integrity of the microorganism'smembrane (cell membrane). Increasing the concentration of solvent in theaqueous phase can impact the membrane's rigidity because the membrane isgenerally oleophillic and includes sterols. The presence of solventadjacent to microorganism can increase the number of holes in the cellmembrane and impact transport of one or more of sugar, glucose, productalcohol, nutrients, and so forth across the cell membrane. Some solventsif present in sufficient concentration in the aqueous phase can impactthe cell membrane's rate of repair. For instance, examples 6A and 6Billustrate how an aromatic hydrocarbon can be made to be biocompatiblewith a proper amount of alkyl substitution. A diisopropyl benzenesolvent with a log P below 6 was found not to be biocompatible. Byadding an additional isopropyl group onto the aromatic ring, theresulting triisopropyl benzene solvent with a log P greater than 6 wasfound to be biocompatible, e.g., biocompatible with a butanologen.

In embodiments, log P of approximately six (6) comprise a log P of 5.5,5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, or 6.3. If log P is much greater thansix (6), in embodiments, a second solvent that is not biocompatible isblended with the base solvent to form a biocompatible binary mixture.Although, a log P of six (6) is described, it is to be appreciated thatdifferent microorganism may have different sensitivities to the presenceof solvent in the aqueous phase, which may also depend on the solvent'sproperties. For example, a second solvent is chosen that exhibits a highproduct alcohol affinity (e.g., a high butanol affinity) in comparisonto the first solvent, but may exhibit low or poorer biocompatibility orhydrophobicity as an indicator of biocompatibility than the firstsolvent. It is to be apparent that multiple solvents can be implementedand/or the solvent mixture is tailored to balance biocompatibility withaffinity towards the fermentation product, and so forth. Otherproperties impacting solvent selection include, but are not limited to,affinity to water, dryness, solubility, its distribution coefficient,reactivity, interfacial tension, viscosity, boiling point, or freezingpoint. Other factors include cost, density, flammability, selectivity tothe fermentation product, thermal stability, and so forth. In examples,much greater than six (6) comprises 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9. In some examples, the second solvent provides exceptionallyhigh butanol affinity but may not be dry. Of interest, surprisingly themoisture content of some solvent mixtures can vary nonlinearly withcomposition and the equilibrium moisture content of any linearcombination of solvents is usually less than the linear combination ofindividual equilibrium solvents moisture content. The log P of linearcombination of solvents can be less or different than the linearcombination of solvent log P values. The Kd of linear combination ofsolvents can be different than the linear combination of Kd values.

In embodiments, the log P for a solvent is calculated from anequilibrium ternary mixture of solvent, octanol, and water as the ratioof the molar concentration of the solvent in the organic phase to themolar concentration of the solvent in the aqueous phase under diluteconditions. In embodiments such as these, five milliliters (5 ml) of azero point zero zero one (0.001) molar solution of the solvent (e.g., afirst or second solvent or a solvent mixture) in octanol is mixed andbrought to phase equilibrium with thirty milliliters (30 ml) of water,sampled and then analyzed.

The log P for a solvent can be determined experimentally or by propertyestimation. The log P of a binary mixture can be measured or calculatedfrom the individual solvent log P values, the molar composition and arigorous non-ideal thermodynamic properties mixing model. In anembodiment, an Aspen Model (Aspen Plus simulation software) wasconstructed that features pure component property estimation by DIPPRand a UNIFAC model for estimating multicomponent phase equilibrium ofmixtures. It is to be understood that “pure component property” refersgenerally to a property of an individual solvent, not necessarily thatthe solvent/component lacks any impurities. For most or all solvents,the log P value calculated by the Aspen Model agrees well withexperimentally determined log P values. Simulations were carried outthat predict the equilibrium molar concentrations of solvent componentsintroduced to the organic and aqueous phases formed from a mixture ofoctanol and water. In addition, the partitioning coefficient ofisobutanol in various solvent mixtures can be estimated by replacingoctanol with isobutanol. In these embodiments, a small amount ofisobutanol is introduced to a mixture of the solvent components andwater. The partitioning coefficient is calculated as the ratio of themass concentration of isobutanol in the equilibrium organic phase to themass concentration of isobutanol in the equilibrium aqueous phase.

Triisopropylbenzene was identified as a base solvent. It has anestimated log P of six point two (6.2). The triisopropylbenzene wastested, and was found to be biocompatible with a butanologen.Triisopropylbenzene has a normal boiling point of two-hundred thirty-sixdegrees Celsius (236° C.) and a density of zero point eight five gramsper cubic centimeter (0.85 g/cm³).

Further, isododecane (2,2,4,6,6-pentamethylheptane) was identified as abase solvent. Isododecane has an estimated log P of six point two (6.2).The isododecane tested was found to be biocompatible with a butanologen.Isododecane has a boiling point of one-hundred seventy-seven degreesCelsius (177° C.) and a density of zero point seven five grams per cubiccentimeter (0.75 g/cm³).

Additionally, isohexadecane (2,2,4,4,6,8,8-heptamethylnonane) wasidentified as a base solvent. Isohexadecane has an estimated log P ofeight point zero (8.0) and is predicted to be biocompatible with abutanologen. Isohexadecane has a boiling point of two-hundred fortydegrees Celsius (240° C.) and a density of zero point seven eight gramsper cubic centimeter (0.78 g/cm³).

In addition, corn oil triglyceride (COTG) was identified as a basesolvent. COTG has an estimated log P of 22.0-24.0 and is predicted to bebiocompatible. It has a density of 0.9 g/cm3.

Tetrabutylurea was also identified as a base solvent. Tetrabutylurea hasan estimated log P of six point six (6.6). Tetrabutylurea was found tobe biocompatible with a butanologen in testing. Tetrabutylurea has aboiling point of three-hundred eighty degrees Celsius (380° C.) and adensity of zero point nine grams per cublic centimeter (0.9 g/cm3).Tetrabutylurea has a high partitioning coefficient for isobutanol. Othershorter chain tetraalkyl ureas that have a log P below six (6) but havea high partitioning coefficient for isobutanol are considered as asecond solvent.

Further, Bisphenol A was identified as a second solvent. Bisphenol A hasan estimated log P of three point four (3.4).

Additionally, DEET (diethyl m-toluamide) was identified as a secondsolvent. DEET has an estimated log P of two point zero (2.0).

In addition, Di-tert-amylphenol was identified as a second solvent.Di-tert-amylphenol has an estimated log P of five point nine (5.9).Other unhindered alkylated phenols can be used as a second solvent. Forexample, di-tert-butylphenol has an estimated log P of 4.9. Oil of thymeand oil of oregano, both isomers of methyl isopropyl phenol, can be usedas a second solvent and are considered environmentally benign. Oil ofthyme and oil of oregano have an estimated log P of three point three(3.3).

Discussion of Sample Solvent Mixture Preparation and Extraction

The following description provides sample techniques, approaches,methods, for selection, preparation, and use of solvent mixtures. Asshould be apparent, the techniques, approaches, methodologies describedherein are applicable to solvents described throughout this disclosure.While the methodologies are described in conjunction with a binarysolvent mixture, multiple solvent mixtures (ternary, quaternary, and soon) can benefit from the techniques described herein.

In embodiments, a method of extracting alcohol from an aqueous solution,such as fermentation broth, comprises selecting which solvents are to beincluded in the solvent mixture. This selection can be based on theindividual solvent's properties (e.g., a property of that solvent).Example properties include, but are not limited to, hydrophobicity (logP), hydrophobicity/log P as an indicator of biocompatibility, Kd,moisture content, and so on. Although the solvents and their respectiveproperties are considered on an individual basis, in some embodimentsselecting includes identifying a solvent with a property that isbeneficial in a particular aspect (relative to the extraction to beperformed) in comparison to another solvent that exhibits that property,but to a lesser extent or exhibits that property negatively (e.g., anunfavorable property). An example of an unfavorable property is asolvent that is highly toxic to a butanologen when butanol is to beextracted from a fermentation broth that includes butanologens. Inanother example, a second solvent is identified that exhibits highbiocompatibility to account for a first solvent that exhibitscomparatively poorer biocompatibility, but also exhibits a beneficialproperty such as high Kd, low moisture, high selectivity to butanol, ifthe solvent mixture is used to extract butanol. Accordingly, thesolvents can be selected so the resultant solvent mixture is generallybalanced, e.g., so the solvent mixture overall exhibits good propertiesrather than exhibiting one beneficial property strongly while exhibitingother relevant properties weakly, poorly, or even negatively.

Moreover, while selection can include considering each solvent and/oreach solvent's properties individually, this can be done within aframework of the other solvents to be included in the solvent mixture.The individual solvent's chemical structures can be considered whendetermining which solvents are to be selected. For example, theheuristic of “like-dissolves-like” can be applied to solvent selectionfrom the solvents identified. Put another way, selection of whichsolvents to include in the solvent mixture can include considering theindividual solvent's chemical structures. Other approaches can be usedas well, e.g., including particular functional groups, chemicalproperties (e.g., para, ortho, meta substituents) and so on. Forexample, two solvents are selected because both have aromatics in theirrespective backbones. Additional examples include selecting two solventas both are aliphatic and branched.

In some embodiments, chemical structure is a threshold criteria that isto be met before solvent properties are considered. In otherembodiments, chemical structure can be considered in parallel withidentifying the solvents and/or the solvent properties. For instance,two solvents are selected to be included in a binary solvent mixturebecause they have generally similar structures, and their respectiveproperties when considered overall are substantially balanced orbalanced. An example of a substantially balanced solvent mixture is twoor more solvents that exhibit “beneficial” strongly or to an acceptableextent while avoiding or exhibiting unfavorable properties to atolerable level. An example of a tolerable level may be a solventmixture that exhibits high Kd, but solvates water (e.g., is moist) to atolerable level for a predetermined set of conditions.

In some implementations, a computing system is configured to selectsolvents by identifying the solvents based on their respective featuresand/or chemical structures. In examples such as these, a computingsystem can be programmed to compare properties associated with varioussolvent on an individual basis to identify solvents that exhibitproperties that benefit the solvent mixture.

The method can further include setting a limit for a ratio of thesolvents to be included in the solvent mixture. For example,hydrophobicity can be used as a limit on a ratio of a first solvent to asecond solvent to determine a ratio limit for a first and a secondsolvent. In the preceding example, hydrophobicity can be used as anindicator or “stand-in” for biocompatibility. Thus, in embodiments, thelimit is set so the ratio of solvents in the solvent mix does not exceedthe limit, thereby being toxic or bio-incompatible with a microorganismproducing a fermentation product, e.g., a product alcohol to beextracted. In some instances, the limit is set so the solvent mixture isonly slightly or minimally toxic, such as to accommodate solvents thatexhibit beneficial features in other aspects. In some instances, thesolvent mixture's hydrophobicity is not indicated by a linearcombination of the hydrophobicities of the solvent mixture's componentsolvents, when taking into account each solvent's mole fraction in thesolvent mixture.

In other words, the hydrophobicity of the solvent mixture (acting as anindicator of biocompatibility) can be used to set a ratio limit for thesolvents to be included in the mixture. For example, a ratio limit ofsolvent A to solvent B is set at log P of six (6) or substantially six(6) so the mixture of solvents A and B is not toxic to butanologenspresent in a fermentation broth. The solvent mixture's hydrophobicity isused as the limit, in some embodiments, because the hydrophobicity ofthe solvent mixture can exhibit a synergistic effect. This is to saythat, hydrophobicity is a property that exhibits a synergistic effect(e.g., an impact in a beneficial way) when solvents A and solvent B aremixed to form the solvent mixture. While a log P of six (6) is describedherein those of skill in the art will appreciate that some strains ofalcohol producing microorganisms exhibit different tolerance levels thatcan be accounted for.

In some embodiments, the method further includes determining a ratio ofthe solvents within the limit. For example, an actual ratio of a firstand second solvent to be included in the solvent mixture is determinedto balance the solvent mixture's overall properties so long as thedetermined ratio is within the limit, i.e., is not toxic to amicroorganism in a fermentation broth. In this general way, the ratio ofthe solvents in the solvent mixture can be tailored to exhibit at leastone synergistic effect so long as the determined ratio is not toxic tothe microorganism. The property that is synergistic may not be indicatedby a linear combination of the solvents that form the solvent mixture.In other words, the extent of a beneficial property of the solventmixture is not indicated (for a two solvent mixture) by a linearcombination of a first and second solvents' respective properties whenconsidering their mole fraction in the solvent mixture. For example, asolvent mixture's Kd is different in a beneficial way than that whichwould be expected based on the Kd for a first and second solvent,respectively. The previous example, generally illustrates a situation inwhich one solvent exhibits a greater impact on the solvent mixture's Kdthan the other solvent's Kd for a two solvent mixture. These principlescan be applied to solvent mixtures with more than two solvents. Intertiary solvent mixtures, the Kd for the individual solvents (forclarity, solvents A, B, and C) can have different impacts. Thus,solvents A's Kd and solvent B's Kd can have a proportionally largerimpact the solvent mixture's Kd than that of solvent C, i.e., solventC's Kd. In other tertiary solvent mixtures, solvent A's moisture canhave a greater impact on the solvent mixture's moisture, than that ofsolvents B and C, when considered individually. Additional examplesincluded, but are not limited to, alcohol selectivity, hydrophobicity asan indicator of biocompatibility, toxicity/biocompatibility (indicateddirectly), and so on.

In embodiments, the selected solvents are combined by mixing thecomponent solvents in the determined ratio so the solvent mixture thatresults is balanced and exhibits at least one synergistic alcoholextraction property that is not indicated by a linear combination of thesolvents' properties and is beneficial for the extraction to beperformed. In additional embodiments, a solvent mixture exhibits morethan one synergistic property.

The solvent mix that results from combining the solvents can becontacted with the aqueous solution to extract alcohol present in thewater into the solvent/organic phase. For example, the solvent mixtureis contacted with fermentation broth that can include, among otherconstituents, water, butanol, butanologen microorganisms, nutrients andso forth.

Optionally, additives are incorporated into the solvent mixture. Exampleadditives include, but are not limited to one or more of antioxidants,antimicrobial agents, additives included to vary the solvent mixture'sproperties (e.g., “salts”) and so forth.

Optionally, in embodiments, the method further comprises maintaining thesolvent mixture so it exhibits predetermined properties. For example,additional solvent mixture is added to adjust its concentration in afermentation system, an additional amount of a component solvent isadded, and so forth.

In embodiments, a method for drying an extractant includes contacting afirst solvent with a fermentation broth to extract alcohol from thebroth. For example, a dry solvent or a solvent mixture is contacted withfermentation broth pumped through an external loop from a fermentor toextract the alcohol. Generally, like that of the other solvents(including a solvent mixture) the first and/or second solvents can beimplemented in a countercurrent extraction configuration. In addition toextracting alcohol, the first solvent can also solubilize at least somewater from the broth before the lean broth is returned to the fermentor.The rich solvent, e.g., extractant including alcohol, now additionallyincludes water. In examples of the present embodiment, the water isremoved by contacting the first solvent that includes the alcohol andwater with a second solvent. This extraction can be performed outsidethe presence of the fermentation broth, as second solvent would likelybe overwhelmed by the water in the fermentation broth. The secondsolvent in these embodiments is a dry solvent that exhibits a highaffinity to water. The second solvent in the previous embodiment may bea hydrophilic solute, such as described above. Glycerol, for example, isused to extract the water from a solvent mixture before the solventmixture including the alcohol is transferred for distillation.Extracting water from rich solvent can avoid drawbacks associated withdistilling in-part water from the product alcohol and solvent. Exampledrawbacks include, but are not limited to increased volume, increasedenergy consumption, distillation/separation considerations, that can beexperience when separating water/solvent/alcohol from one another.Removing water from rich solvent may be performed because the solvent islikely reused for subsequent extractions.

As will be appreciated by those of skill in the art, the solvent dryingmethod, techniques and approaches can be implemented with a solventmixture, this is to say that the first solvent comprises the solventmixture as described throughout this document. While a variety of thefirst and second solvent's properties can be considered when determiningwhich solvent to use as the drying or second solvent, these propertiesgenerally mirror those considered with respect to the first and secondsolvents in a solvent mixture.

Further Discussion of Examples and Individual Solvent's Impact onSolvent Mixture

The following discussion is provided to further describe individualsolvent characteristics. The individual solvent's properties can be usedto tailoring solvent mixture by selecting solvents that exhibitproperties that are beneficial for butanol extraction but are notindicated by a liner combination of the first and second solvent's,respective, properties relative to that solvent's mole fraction in thesolvent mixture.

Referring now to FIG. 11A, a graphic representation illustrating how anindividual solvent can exhibit a beneficial property is discussed. Thisgraphical representation was generated using Aspen modeling software asis described at various locations in this document. In this embodiment,isododecane is added to COFA to improve the solvent mixture's dryness.For example, adding isododecane to COFA even in a small amount canreduce the moisture content of the solvent mixture. Accordingly,adjusting the molar concentration of COFA to isododecane impacts themoisture content of the resultant solvent mixture in a non-linearmanner, e.g., synergistically in a favorable way for the extraction tobe performed. As is illustrated, the dashed line illustrates how COFAand isododecane are anticipated to behave with respect to moisturecontent based on a linear combination of the two, while the solid(curved) line indicates how increasing the concentration of isododecanein the solvent mixture impacts moisture content of the solvent mixture,up to a solvent mixture that is one hundered percent (100%) isododecaneon a molar basis. As can be seen, increasing the concentration ofisododecane up to approximately seventy percent (70%) can improve thedryness of the solvent mixture (COFA and isododecane) over that of COFAalone for use in extracting alcohol from fermentation broth. Whileisododecane concentrations up to seventy percent (70%) by molarconcentration show lower moisture content (non-linear behavior), it isto be appreciated that isododecane can be implemented at lowerconcentrations.

Referring now to FIGS. 11B and 11C, these figures illustrate Kd incomparison to different molar concentrations to illustrate the impact oftwo similar solvents (isododecane and isododecanol). The mixtures ofexample 11B exhibit a butanol affinity that is higher than what would beexpected from a linear combination. Solvent mixtures of isododecane andisododecanol in some examples behave less like a linear combination thatthat of other solvent mixtures. For example, a solvent mixtures oftetrabutylurea with isododecane illustrated in 11 C can behavesubstantially like a linear combination while, comparatively,isododecane and isododecanol behave in a less or non-ideal manner withrespect to Kd.

Referring to FIG. 12, in embodiments, some solvents do not exhibitexpected tradeoffs between, for example, Kd and hydrobhobicity. Forexamples, alkylphenols and alkyl ureas do not follow an expectedtradeoff between Kd and log P for butanol extraction. This is to saythat these solvents do not exhibit a drop-off in butanol distributionequilibrium partitioning in comparison to that solvent's hydrophobicityas is generally expected for organic solvents. The line (e.g., the curvecaptioned “general tradeoff”), was graphically determined using standardtechniques from data points of solvents that do exhibit this correlationbetween log P and the various solvent's butanol partition coefficient ascan be observed. As also can be seen, thymol, tributylphosphate, andtetrabutylurea exhibit high butanol distribution equilibriumpartitioning between the aqueous and solvent phases, while having higherlog P than that which is expected. Accordingly, inclusion of one or moreof these solvents in a solvent mixture, even at low concentration, canincrease the mixture's butanol affinity beyond that indicated by alinear combination of one of these solvents and a co-solvent (secondsolvent). Similar examples exist for toxicity (biocompatibility) (pleasesee FIG. 12), and so forth.

Referring now to FIG. 13, this figure is an illustration of boilingpoints in comparison to log P for a variety of solvents. As shown,different solvents exhibit different boiling points/hydrophobicitieseven though they have the same number of carbons atoms. Thesedifferences in log P to boiling point can be used to tailor solventmixture properties to optimize beneficial properties while minimizing oreliminating unfavorable properties. The differences in boiling pointsand/or hydrophobicities may be attributable to the solvents' structure,including but not limited to, branched, aromatic, orientation ofsubstituent groups, aliphatic, and so forth. Of note, thymol exhibits alow boiling point and low toxicity as indicated by its boiling point ofapproximately two-hundred thirty degrees Celsius (230° C.) and its log Pof approximately three point three (3.3). In addition, thymol isgenerally considered environmentally benign and is readily available asit is prevalent in thyme.

Referring now to FIG. 14, this figure is a graphic illustration ofdifferent molar concentrations of thymol in respectively corn oil and1,3,5-triisopropylbenzene. As can be observed, when mixed in a twosolvent system with corn oil, the log P of corn oil/thymol decreasesfrom a high log P of approximately eighteen (19) to a log P ofapproximately six (6) with the addition of approximately one molarpercent thymol, indicating a non-linear relationship in a disfavorablemanner. Although thymol exhibits high butanol distribution equilibriumpartitioning while having a higher log P than that expected for itschemical structure, some experiments indicate a mixture of thymol/cornoil exhibit poor overall properties as the Kd for the thymol/corn oilmixture drops to approximately a log P of four (4). Some potentialstructural influences include, but are not limited to, differences inchemical structure between corn oil and and thymol (e.g., linear versusaromatic with ortho/para substituents) and the like. Generally,structurally dissimilar solvents are considered to be disadvantaged asthey do not follow a like-like heuristic.

In contrast, a solvent mixture of 1,3,5-triisopropylbenzene and thymolalthough structurally similar (both include aromatics and propyl groups)showed relatively little change in log P when the molar concentration ofthymol is increased (up to 10% thymol is illustrated). This is to saythat 1,3,5-triisopropylbenzene and thymol behaved in a linear fashion,but in a favorable way. Thus, while exhibiting low log P individually,thymol when combined with triisopropylbenzene does not show marked log Pchange shown by the corn oil/thymol mixture that does not follow thelike-dissolves-like rubric. The behavior of thymol and1,3,5-triisopropylbenzene may be because both include aromatics and havea propyl group.

Further modifications and alternative embodiments of this disclosurewill be apparent to those skilled in the art in view of thisdescription. At times methods are described that can be implemented inconjunction with a computing system configured to perform the method orat least a portion of the method. In situations such as this, acomputing system can be a general purpose computer that is programed toperform the method or step. It is to be apparent that the method can beimplemented as a set of instructions embodied in computer readablemedia, e.g., tangible, non-transitory media. Further, computing systemsin accordance with the present disclosure can provide output in avariety of ways including displaying information, being configured tocontrol equipment (e.g., fermentation or extraction devices in aparticular manner, and so on). It will be recognized, therefore, thatthe present invention is not limited by these example arrangementsand/or hardware in the computing system. Accordingly, this descriptionis to be construed as illustrative only and is for the purpose ofteaching those skilled in the art the manner of carrying out themethods, approaches, devices, equipment, systems, and so forth. It is tobe understood that the forms of the invention herein shown and describedare to be taken as the presently preferred embodiments. Various changesmay be made in the shape, size and arrangement of parts. For example,equivalent elements may be substituted for those illustrated anddescribed herein, and certain features of the invention may be utilizedindependently of the use of other features, all as would be apparent toone skilled in the art after having the benefit of this description.

What is claimed is:
 1. A method for recovering butanol from afermentation medium, the method comprising: a) providing a fermentationmedium comprising butanol, water, and a recombinant microorganismcomprising a butanol biosynthetic pathway, wherein the recombinantmicroorganism produces butanol; b) contacting the fermentation mediumwith a water immiscible organic extractant composition comprising a drysolvent and a second solvent to form a butanol-containing organic phaseand an aqueous phase, wherein the dry solvent is a saturated hydrocarbonthat is a branched C7 to C22 alkane or a mixture thereof; and c)recovering the butanol from the butanol-containing organic phase.
 2. Themethod of claim 1, wherein the C7 to C22 alkane is a derivative ofisobutanol, wherein the derivative of isobutanol is triisobutylene,diisobutylene, tetraisobutylene, isooctane, isohexadecane, orisododecane.
 3. The method of claim 2, wherein the derivative ofisobutanol is isododecane.
 4. The method of claim 1, wherein thecontacting of the organic extractant composition with the fermentationmedium occurs in a fermentor.
 5. The method of claim 1, furthercomprising transferring a portion of the fermentation medium from afermentor to a vessel, wherein the contacting of the organic extractantcomposition with the fermentation medium occurs in the vessel.
 6. Themethod of claim 1, wherein the second solvent is a C4 to C22 fattyalcohol, a C4 to C28 fatty acid, an ester of a C4 to C28 fatty acid, aC4 to C22 fatty aldehyde, a C7 to C22 ether, a phosphate, an amide, aurea, a phenol, a phosphinate, a carbamate, a phosphoramide, a phosphineoxide, a salicylate, a paraben, or mixtures thereof.
 7. The method ofclaim 6, wherein the second solvent increases a butanol partitioncoefficient of the organic extractant composition.
 8. The method ofclaim 1, wherein the second solvent is oleyl alcohol, behenyl alcohol,cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleaicacid, lauric acid, myristic acid, stearic acid, octanoic acid, decanoicacid, undecanoic acid, methyl myristate, methyl oleate, 1-nonanol,1-decanol, 2-undecanol, 1-nonanal, undecanol, undecanal, isododecanol,2,6,8-trimethyl-4-nonanol, lauric aldehyde, 2-methylundecanal, oleamide,linoleamide, palmitamide, steaylamide, 2-ethyl-1-hexanol,2-hexyl-1-decanol, 2-octyl-1-dodecanol,3,4,5,6,6-pentamethyl-2-heptanol, or mixtures thereof.
 9. The method ofclaim 8, wherein the second solvent increases a butanol partitioncoefficient of the organic extractant composition.
 10. The method ofclaim 1, wherein the recovered butanol has an effective titer from about30 grams per liter to about 80 grams per liter of the fermentationmedium.
 11. The method of claim 1, wherein the recovered butanol has aneffective titer of at least 50 grams per liter of the fermentationmedium.
 12. The method of claim 1, wherein the butanol is isobutanol.13. A composition comprising butanol in a water immiscible organicextractant composition, wherein the organic extractant compositioncomprises a dry solvent and a second solvent, wherein the dry solvent isa saturated hydrocarbon that is a branched C7 to C22 alkane or a mixturethereof.
 14. The composition of claim 13, wherein the C7 to C22 alkaneis a derivative of isobutanol, wherein the derivative of isobutanol istriisobutylene, diisobutylene, tetraisobutylene, isooctane,isohexadecane, or isododecane.
 15. The composition of claim 13, whereinthe derivative of isobutanol is isododecane.
 16. The composition ofclaim 13, wherein the second solvent is a C4 to C22 fatty alcohol, a C4to C28 fatty acid, an ester of a C4 to C28 fatty acid, a C4 to C22 fattyaldehyde, a C7 to C22 ether, a phosphate ester, an amide, a urea, aphenol, a phosphinate, a carbamate, a phosphoramide, a phosphine oxide,a salicylate, a paraben, or mixtures thereof.
 17. The composition ofclaim 13, wherein the second solvent is oleyl alcohol, behenyl alcohol,cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleaicacid, lauric acid, myristic acid, stearic acid, octanoic acid, decanoicacid, undecanoic acid, methyl myristate, methyl oleate, 1-nonanol,1-decanol, 2-undecanol, 1-nonanal, undecanol, undecanal, isododecanol,2,6,8-trimethyl-4-nonanol, lauric aldehyde, 2-methylundecanal, oleamide,linoleamide, palmitamide, steaylamide, 2-ethyl-1-hexanol,2-hexyl-1-decanol, 2-octyl-1-dodecanol,3,4,5,6,6-pentamethyl-2-heptanol, or mixtures thereof.
 18. Thecomposition of claim 13, wherein the butanol is isobutanol.